Recombinant retroviruses

ABSTRACT

Recombinant retroviruses carrying a vector construct capable of preventing, inhibiting, stabilizing or reversing infectious, cancerous or auto-immune diseases are disclosed. More specifically, the recombinant retroviruses of the present invention are useful for (a) stimulating a specific immune response to an antigen or a pathogenic antigen; (b) inhibiting a function of a pathogenic agent, such as a virus; and (c) inhibiting the interaction of an agent with a host cell receptor. In addition, eucaryotic cells infected with, and pharmaceutical compositions containing such a recombinant retrovirus are disclosed. Various methods for producing recombinant retroviruses having unique characteristics, and methods for producing transgenic packaging animals or insects are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.08/136,739, filed Oct. 12, 1993, U.S. Pat. No. 5,716,826; which is acontinuation of U.S. patent application Ser. No. 07/395,932, filed Aug.18, 1989, now abandoned; which is a continuation-in-part of U.S. patentapplication Ser. No. 07/170,515, filed Mar. 21, 1988, now abandoned.

TECHNICAL FIELD

The present invention relates generally to retroviruses, and morespecifically, to recombinant retroviruses which are capable ofdelivering vector constructs to susceptible target cells. These vectorconstructs are typically designed to express desired proteins in targetcells, for example, proteins which stimulate immunogenic activity orwhich are conditionally active in defined cellular environments.

BACKGROUND OF THE INVENTION

Although bacterial diseases are, in general, easily treatable withantibiotics, very few effective treatments or prophylactic measuresexist for many viral, cancerous, and other nonbacterial diseases,including genetic diseases. Traditional attempts to treat these diseaseshave employed the use of chemical drugs. In general, these drugs havelacked specificity, exhibited high overall toxicity, and thus have beentherapeutically ineffective.

Another classic technique for treating a number of nonbacterial diseasesinvolves the elicitation of an immune response to a pathogenic agent,such as a virus, through the administration of a noninfectious form ofthe agent, such as a killed virus, thereby providing antigens from thepathogenic agent which would act as an immunostimulant.

A more recent approach for treating viral diseases, such as acquiredimmunodeficiency syndrome (AIDS) and related disorders, involvesblocking receptors on cells susceptible to infection by HIV fromreceiving or forming a complex with viral envelope proteins. Forexample, Lifson et al. (Science 232:1123-1127, 1986) demonstrated thatantibodies to CD4 (T4) receptors inhibited cell fusion (syncytia)between infected and noninfected CD4 presenting cells in vitro. Asimilar CD4 blocking effect using monoclonal antibodies has beensuggested by McDougal et al. (Science 231:382-385, 1986). Alternatively,Pert et al. (Proc. Natl. Acad. Sci. USA 83:9254-9258, 1986) havereported the use of synthetic peptides to bind T4 receptors and blockHIV infection of human T-cells, while Lifson et al. (J. Exp. Med.164:2101, 1986) have reported blocking both syncytia and virus/T4 cellfusion by using a lectin which interacts with a viral envelopeglycoprotein, thereby blocking it from being received by CD4 receptors.

A fourth, recently suggested technique for inhibiting a pathogenicagent, such as a virus, which transcribes RNA is to provide antisenseRNA which complements at least a portion of the transcribed RNA, andbinds thereto, so as to inhibit translation (To et al., Mol. Cell. Biol.6:758, 1986).

However, a major shortcoming of the techniques described above is thatthey do not readily lend themselves to control as to the time, locationor extent to which the drug, antigen, blocking agent or antisense RNAare utilized. In particular, since the above techniques requireexogenous application of the treatment agent (i.e., exogenous to thesample in an in vitro situation), they are not directly responsive tothe presence of the pathogenic agent. For example, it may be desirableto have an immunostimulant expressed in increased amounts immediatelyfollowing infection by the pathogenic agent. In addition, in the case ofantisense RNA, large amounts would be required for useful therapy in ananimal, which under current techniques would be administered withoutregard to the location at which it is actually needed, that is, at thecells infected by the pathogenic agent.

As an alternative to exogenous application, techniques have beensuggested for producing treatment agents endogenously. Morespecifically, proteins expressed from viral vectors based on DNAviruses, such as adenovirus, simian virus 40, bovine papilloma, andvaccinia viruses, have been investigated. By way of example, Panicali etal. (Proc. Natl. Acad. Sci. USA 80:5364, 1983) introduced influenzavirus hemagglutinin and hepatitis B surface antigens into the vacciniagenome and infected animals with the virus particles produced from suchrecombinant genes. Following infection, the animals acquired immunity toboth the vaccinia virus and the hepatitis B antigen.

However, a number of difficulties have been experienced to date withviral vectors based on DNA viruses. These difficulties include (a) theproduction of other viral proteins which may lead to pathogenesis or thesuppression of the desired protein; (b) the capacity of the vector touncontrollably replicate in the host, and the pathogenic effect of suchuncontrolled replication; (c) the presence of wild-type virus which maylead to viremia; and (d) the transitory nature of expression in thesesystems. These difficulties have virtually precluded the use of viralvectors based on DNA viruses in the treatment of viral, cancerous, andother nonbacterial diseases, including genetic diseases.

Due to the nontransitory nature of their expression in infected targetcells, retroviruses have been suggested as a useful vehicle for thetreatment of genetic diseases (for example, see F. Ledley, The Journalof Pediatrics 110:1, 1987). However, in view of a number of problems,the use of retroviruses in the treatment of genetic diseases has notbeen attempted. Such problems relate to (a) the apparent need to infecta large number of cells in inaccessible tissues (e.g., brain); (b) theneed to cause these vectors to express in a very controlled andpermanent fashion; (c) the lack of cloned genes; (d) the irreversibledamage to tissue and organs due to metabolic abnormalities; and (e) theavailability of other partially effective therapies in certaininstances.

In addition to genetic diseases, other researchers have contemplatedusing retroviral vectors to treat nongenetic diseases (see, for example,EP 243,204--Cetus Corporation; Sanford, J. Theor. Biol. 130:469, 1988;Tellier et al., Nature 318:414, 1985; and Bolognesi et al., Cancer Res.45:4700, 1985).

Tellier et al. suggested protecting T-cell clones by apparentlyinfecting stem cells with "defective" HIV having a genome which couldexpress antisense RNA to HIV RNA. Bolognesi et al. have suggested theconcept of generating a nonvirulent HIV strain to infect stem cells sothat T4 cells generated therefrom would carry interfering, nonvirulentforms of virus and thereby protect those cells from infection byvirulent HIV. However, it would appear that the "attenuated" or"defective" HIV viruses used in both of the foregoing papers couldreproduce (i.e., are not replication defective) such that the resultingviruses could infect other cells, with the possibility of an increasedrisk of recombination with previously present HIV or other sequences,leading to loss of attenuation. Non-nonreplicative forms wouldnecessitate a defective helper or packaging line for HIV. However, sincethe control of HIV gene expression is complex, such cells have to datenot been constructed. Furthermore, as the infecting attenuated ordefective virus is not chimeric (a "nonchimeric" retrovirus being onewith substantially all of its vector from the same retrovirus species),even if they were made replication defective, for example, by deletionfrom their genomes of an essential element, there still exists asignificant possibility for recombination within the host cells withresultant production of infectious viral particles.

Although Sanford (J. Theor. Biol. 130:469, 1988) has also proposed usinga genetic cure for HIV, he notes that due to the potential that existsfor creating novel virulent viruses via genetic recombination betweennatural AIDS virus and therapeutic retroviral vectors carrying anti-HIVgenes, retroviral gene therapy for AIDS may not be practical. Similarly,while McCormick & Kriegler (EP 243,204 A2) have proposed usingretroviral vectors to deliver genes for proteins, such as tumor necrosisfactor (TNF), the techniques they describe suffer from a number fdisadvantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides recombinant retrovirusescarrying a vector construct capable of preventing, inhibiting,stabilizing or reversing infectious, cancerous, auto-immune or immunediseases. Such diseases include HIV infection, melanoma, diabetes, graftvs. host disease, Alzheimer's disease, and heart disease.

The present invention is directed, in part, toward methods for (a)stimulating a specific immune response, either humoral or cell-mediated,to an antigen or pathogenic antigen; (b) inhibiting a function of apathogenic agent, such as a virus; and (c) inhibiting the interaction ofan agent with a host cell receptor, through the use of recombinantretroviruses.

More specifically, within one aspect of the present invention, a methodfor stimulating a specific immune response is provided, comprisinginfecting susceptible target cells with recombinant retrovirusescarrying a vector construct that directs the expression of an antigen ormodified form thereof in infected target cells. For purposes of thepresent invention, the term "infecting" includes the introduction of DNAsequences through viral vectors, transfection or other means, such asmicroinjection, protoplast fusion, etc. Where an immune response is tobe stimulated to a pathogenic antigen, the recombinant retrovirus ispreferably designed to express a modified form of the antigen which willstimulate an immune response and which has reduced pathogenicityrelative to the native antigen. This immune response is achieved whencells present antigens in the correct manner, i.e., in the context ofthe MHC class I and/or II molecules along with accessory molecules suchas CD3, ICAM-1, ICAM-2, LFA-1, or analogs thereof (e.g., Altmann et al.,Nature 338:512, 1989). Cells infected with retroviral vectors areexpected to do this efficiently because they closely mimic genuine viralinfection.

This aspect of the invention has a further advantage over other systemsthat might be expected to function in a similar manner, in that thepresenter cells are fully viable and healthy, and no other viralantigens (which may well be immunodominant) are expressed. This presentsa distinct advantage since the antigenic epitopes expressed can bealtered by selective cloning of sub-fragments of the gene for theantigen into the recombinant retrovirus, leading to responses againstimmunogenic epitopes which may otherwise be overshadowed byimmunodominant epitopes. Such an approach may be extended to theexpression of a peptide having multiple epitopes, one or more of theepitopes derived from different proteins. In addition, the presentinvention provides for a more efficient presentation of antigens throughthe augmentation or modification of the expression of presentingaccessory proteins (e.g., MHC I, ICAM-1, etc.) in antigen presentingcells.

An immune response can also be achieved by transferring to anappropriate immune cell (such as a T lymphocyte) the gene for thespecific T-cell receptor which recognizes the antigen of interest (inthe context of an appropriate MHC molecule if necessary), for animmunoglobulin which recognizes the antigen of interest, or for a hybridof the two which provides a CTL response in the absence of the MHCcontext.

In the particular cases of disease caused by HIV infection, whereimmunostimulation is desired, the antigen generated from the recombinantretroviral genome is of a form which will elicit either or both an HLAclass I- or class II-restricted immune response. In the case of HIVenvelope antigen, for example, the antigen is preferably selected fromgp 160, gp 120, and gp 41, which have been modified to reduce theirpathogenicity. In particular, the antigen selected is modified to reducethe possibility of syncytia, to avoid expression of epitopes leading toa disease enhancing immune response, to remove immunodominant, butstrain-specific epitopes or to present several strain-specific epitopes,and allow a response capable of eliminating cells infected with most orall strains of HIV. Antigens from other HIV genes, such as gag, pol,vif, nef, etc., may also provide protection in particular cases.

In another aspect of the present invention, methods for inhibiting afunction of a pathogenic agent necessary for disease, such as diseasescaused by viral infections, cancers or immunological abnormalities, aredisclosed. Where the pathogenic agent is a virus, the inhibited functionmay be selected from the group consisting of adsorption, replication,gene expression, assembly, and exit of the virus from infected cells.Where the pathogenic agent is a cancerous cell or cancer-promotinggrowth factor, the inhibited function may be selected from the groupconsisting of viability, cell replication, altered susceptibility toexternal signals, and lack of production of anti-oncogenes or productionof mutated forms of anti-oncogenes. Such inhibition may be providedthrough recombinant retroviruses carrying a vector construct encoding"inhibitor palliatives," such as: (a) antisense RNA; (b) a mutantprotein analogue to a pathogenic protein, which interferes withexpression of the pathogenic state; (c) a protein that activates anotherwise inactive precursor; (d) defective interfering structuralproteins; (e) peptide inhibitors of viral proteases or enzymes; (f)tumor suppressor genes; or (g) a RNA ribozyme capable of specificallycutting and degrading RNA molecules corresponding to the pathogenicstate. Alternatively, such inhibition is attained by a recombinantretrovirus capable of site-specific integration into pathogenic genes,thereby disrupting them.

Such inhibition may also be accomplished through the expression of apalliative that is toxic for a diseased cell. Where a toxic palliativeis to be produced by cells containing the recombinant viral genome, itis important that either the recombinant retrovirus infect only targetcells or express the palliative only in target cells, or both. In eithercase, the final toxic agent is localized to cells in the pathogenicstate. Where expression is targeted, the pathogenic agent controllingexpression of the toxic palliative could be, for instance, a proteinproduced through transcription and translation of a pathogenic viralgenome present in the cell.

It should be understood in the foregoing discussion, and throughout thisapplication, that when reference is made to the viral construct"expressing" or "producing" any substance in a cell, or the like, thisin fact refers to the action of the resulting provirus following reversetranscription of the viral RNA in the cell. In the context of a toxicpalliative, the consequent killing effect may not necessarily requirethe permanent integration of the recombinant viral genome into the hostgenome, but simply a reasonably long-term expression of a toxicpalliative gene, in whatever form desirable, over a reasonably longperiod of time (several days to one month). Thus, other nonintegratingviral vectors such as, but not limited to, adenoviral vectors may beused for this purpose. Examples of conditional toxic palliatives includerecombinant retroviruses encoding (a) a toxic gene product under thecontrol of a cell cycle-specific promoter, a tissue-specific promoter orboth; (b) a gene product which is conditionally expressed and which initself is not toxic but which processes within target cells a compoundor drug from a nontoxic precursor form to an active toxic form; (c) agene product which is not in itself toxic, but when processed by aprotein, such as protease specific to a viral or other pathogen, isconverted into a toxic form; (d) a conditionally expressed reporter geneproduct on the cell surface which identifies the pathogenic cells forattack, for example, by immunotoxins; (e) conditionally expressed geneproducts on the cell surface which lead to a toxic effect by interactionwith extracellular factors; and (f) conditionally expressed ribozymesspecific for RNA molecules essential for viability.

Within a related aspect, the present invention also provides methods fordiminishing or eliminating an unwanted or deleterious immune response.Immune suppression, where appropriate, can be achieved by targetingexpression of immune suppressive genes, such as the virally derived E3gene of adenovirus.

Within another aspect of the present invention, methods are disclosedfor inhibiting the interaction of viral particles with cells, cells withcells, or cells with factors. The methods generally comprise infectingsusceptible cells with a recombinant, replication defective retroviruswhich directs the expression of a blocking element in infected cells,the blocking element being capable of binding with a cell receptor(preferably the host cell receptor) either while the receptor isintracellular or on the cell surface, or alternatively, by binding withthe agent. In either event, the interaction is blocked.

Regardless of the means by which the recombinant retrovirus exerts itsimmunogenic or inhibitory action as described above, it is preferredthat the retroviral genome be "replication defective" (i.e., ircapableof reproducing in cells infected with it). Thus, there will be only asingle stage of infection in either an in vitro or in vivo application,thereby substantially reducing the possibility of insertionalmutagenesis. Preferably, to assist in this end, the recombinantretrovirus lacks at least one of the gag, pol, or env genes. Further,the recombinant viral vector is preferably chimeric (that is, the genewhich is to produce the desired result is from a different source thanthe remainder of the retrovirus). A chimeric construction furtherreduces the possibility of recombination events within cells infectedwith the recombinant retrovirus, which could produce a genome that cangenerate viral particles.

Within another aspect of the present invention, recombinant retroviruseswhich are useful in executing the above methods as well as deliveringother therapeutic genes are disclosed. The present invention alsoprovides a method for producing such recombinant retroviruses in whichthe retroviral genome is packaged in a capsid and envelope, preferablythrough the use of a packaging cell. The packaging cells are providedwith viral protein-coding sequences, preferably in the form of twoplasmids, which produce all proteins necessary for production of viableretroviral particles, an RNA viral construct which will carry thedesired gene, along with a packaging signal which will direct packagingof the RNA into the retroviral particles.

The present invention additionally provides a number of techniques forproducing recombinant retroviruses which can facilitate:

i) the production of higher titres from packaging cells;

ii) packaging of vector constructs by means not involving the use ofpackaging cells;

iii) the production of recombinant retroviruses which can be targetedfor preselected cell lines; and

iv) the integration of the proviral construct into a preselected site orsites in a cell's genome.

One technique for producing higher titres from packaging cells takesadvantage of the discovery that of the many factors which can limittitre from a packaging cell, one of the most limiting is the level ofexpression of the packaging proteins, namely, the gag, pol, and envproteins, as well as the level of expression of the retroviral vectorRNA from the proviral vector. This technique allows the selection ofpackaging cells which have higher levels of expression (i.e., producehigher concentrations) of the foregoing packaging proteins and vectorconstruct RNA. More specifically, this technique allows selection ofpackaging cells which produce high levels of what is referred to hereinas a "primary agent," which is either a packaging protein (e.g., gag,pol, or env proteins) or a gene of interest to be carried into thegenome of target cells (typically as a vector construct). This isaccomplished by providing in packaging cells a genome carrying a gene(the "primary gene") which expresses the primary agent in the packagingcells, along with a selectable gene, preferably downstream from theprimary gene. The selectable gene expresses a selectable protein in thepackaging cells, preferably one which conveys resistance to an otherwisecytotoxic drug. The cells are then exposed to a selecting agent,preferably the cytotoxic drug, which enables identification of thosecells which express the selectable protein at a critical level (i.e., inthe case of a cytotoxic drug, by killing those cells which do notproduce a level of resistance protein required for survival).

Preferably, in the technique briefly described above, the expressions ofboth the selectable and primary genes is controlled by the samepromoter. In this regard, it may be preferable to utilize a retroviral5' LTR. In order to maximize titre of a recombinant retrovirus frompackaging cells, this technique is first used to select packaging cellsexpressing high levels of all the required packaging proteins, and thenis used to select which of these cells, following transfection with thedesired proviral construct, produce the highest titres of therecombinant retrovirus.

Techniques are also provided for packaging of vector constructs by meansnot involving the use of packaging cells. These techniques make use ofother vector systems based on viruses such as other unrelatedretroviruses, baculovirus, adenovirus, or vaccinia virus, preferablyadenovirus. These viruses are known to express relatively high levels ofproteins from exogenous genes provided therein. For such DNA virusvectors, recombinant DNA viruses can be produced by in vivorecombination in tissue culture between viral DNA and plasmids carryingretroviral or retroviral vector genes. The resultant DNA viral vectorscarrying either sequences coding for retroviral proteins or forretroviral vector RNA are purified into high titre stocks.Alternatively, the constructs can be constructed in vitro andsubsequently transfected into cells which provide in trans viralfunctions missing from the DNA vectors. Regardless of the method ofproduction, high titre (10⁷ to 10¹¹ units/ml) stocks can be preparedthat will, upon infection of susceptible cells, cause high levelexpression of retroviral proteins (such as gag, pol, and env) or RNAretroviral vector genomes, or both. Infection of cells in culture withthese stocks, singly or in combination, will lead to high-levelproduction of retroviral vectors, if the stocks carry the viral proteinand viral vector genes. This technique, when used with adenovirus orother mammalian vectors, allows the use of primary cells (e.g., fromtissue explants or cells such as WI38 used in production of vaccines) toproduce recombinant retroviral vectors.

In an alternative to the foregoing technique, recombinant retrovirusesare produced by first generating the gag/pol and env proteins from acell line infected with the appropriate recombinant DNA virus in amanner similar to the preceding techniques, except that the cell line isnot infected with a DNA virus carrying the vector construct.Subsequently, the proteins are purified and contacted with the desiredviral vector RNA made in vitro, transfer RNA (tRNA), liposomes, and acell extract to process the env protein into the liposomes, such thatrecombinant retroviruses carrying the viral vector RNA are produced.Within this technique, it may be necessary to process the env proteininto the liposomes prior to contacting them with the remainder of theforegoing mixture. The gag/pol and env proteins may also be made afterplasmid mediated transfection in eukaryotic cells, in yeast, or inbacteria.

The technique for producing recombinant retroviruses which can betargeted for preselected cell lines utilizes recombinant retroviruseshaving one or more of the following: an env gene comprised of acytoplasmic segment of a first retroviral phenotype, and anextracellular binding segment exogenous to the first retroviralphenotype (this binding segment is from a second viral phenotype or fromanother protein with desired binding properties which is selected to beexpressed as a peptide which will bind to the desired target); anotherviral envelope protein; another ligand molecule in place of the normalenvelope protein; or another ligand molecule along with an envelopeprotein that does not lead to infection of the target cell type.

Techniques for integrating a retroviral genome at a specific site in theDNA of a target cell involve the use of homologous recombination, oralternatively, the use of a modified integrase enzyme which willrecognize a specific site on the target cell genome. Such site-specificinsertion allows genes to be inserted at sites on the target cells' DNA,which will minimize the chances of insertional mutagenesis, minimizeinterference from other sequences on the DNA, and allow insertion ofsequences at specific target sites so as to reduce or eliminate theexpression of an undesirable gene (such as a viral gene) in the DNA ofthe target cell.

It will be appreciated that any of the above-described techniques may beused independently of the others in particular situations, or can beused in conjunction with one or more of the remainder of the techniques.The present invention is also directed to a pharmaceutical compositioncomprising a recombinant virus as disclosed herein in combination with apharmaceutically acceptable carrier or diluent.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts three different families of vectors used to produce HIVenv and which may or may not have the selectable SV-Neo cassetteinserted.

FIG. 2 illustrates the HIV env expression levels seen in polyacrylamidegel electrophoresis of HIV env-specific radioimmune precipitations ofextracts of human Sup Ti cells transfected with the vectors shown. Themarkers are in kilodaltons, gp 160 and gp 120 mark the appropriateproteins, and 517+tat is the positive control (HIV LTR driving env inthe presence of tat).

FIG. 3 depicts the protocol for testing T-cell killing induced in miceinjected with syngeneic tumor cells expressing HIV env (the vector ispAF/Env^(r) /SV₂ neo).

FIG. 4A-1 graphically depicts the results of the experimental protocolof FIG. 3 showing specific killing of BC10MEEenv-29 target cells.

FIG. 4A-2 graphically depicts the results of the experimental protocolof FIG. 3 showing resistance to killing in BC10ME control cells.

FIG. 4B illustrates the specificity of the CTL for HIV envelopeantigens.

FIG. 4C demonstrates the phenotype of the effector cell populationgenerated in the experimental protocol in FIG. 3. The effector cellpopulation is that of an L3T4⁻ lyt2⁺ (CD4⁻ CD8⁺) T lymphocyte.

FIG. 4D illustrates the MHC restriction requirements for the Balb/canti-BCenv CTL response.

FIG. 4E demonstrates that CTL can be induced in vivo by irradiatednonproliferating stimulator cells.

FIG. 4F-1 illustrates the dose-response relationship of immunizingBalb/c mice with BCenv stimulator cells.

FIG. 4F-2 illustrates the-dose-response relationship of immunizingBalb/c mice with BC target cells.

FIG. 4G-1 demonstrates the generation of CTL responses by differentH-2^(d) mouse strains as well as F1 hybrid mice against BCenv targetcells.

FIG. 4G-2 demonstrates the generation of CTL responses by differentH-2^(d) mouse strains as well as F1 hybrid mice against BC target cells.

FIG. 5 depicts a vector designed to express sCD4.

FIG. 6 illustrates the construction of the plasmids carrying the vectorsTK1 (without SV-Neo) and TK3 (plus SV-Neo).

FIG. 7 illustrates the construction of the plasmid carrying the vectorKTVIHAX.

FIG. 8 illustrates the construction of the plasmids carrying the vectorsKTVIH5 (without SV-Neo) and KTVIH Neo (with SV-Neo).

FIG. 9 illustrates construction of the plasmid carrying the vectorMHMTK-Neo.

FIG. 10 illustrates the construction of the plasmid carrying the vectorRRKTVIH.

FIG. 11 illustrates the construction of the plasmids carrying thetat-his (tat in sense direction) or αtat (tat in antisense direction)vectors.

FIG. 12 graphically depicts the preferential killing of PA317 cellsinfected with tathis vector (5 clones, TH1-5) compared to control PA317,upon infection with the three conditional lethal vectors shown andtreatment with acyclovir (ACV).

FIG. 13 illustrates the constuction of the plasmid carrying the vector4TVIHAX.

FIG. 14 depicts the construction of a viral vector carrying HIVinducible marker/reporter genes such as alkaline phosphatase (AP).

FIG. 15 depicts the structure of an HIV inducible marker/reporter genecarried on a plasmid which can be transfected into cells.

FIG. 16 graphically depicts a time course of HIV infection of Sup T1cells carrying the AP marker in FIG. 15 with HIV at variousconcentrations of AZT. The level of HIV infection was measured by takingsmall aliquots of supernatant.

FIG. 17 graphically depicts the results of the same experiment as inFIG. 16, but with ddC as the HIV inhibitor.

FIG. 18 diagrammatically illustrates the number of cells surviving afterphleomycin selection upon transfection of cells with a plasmid whichexpresses the phlemoycin resistance gene (PRG) directly from a promoter(right, complete line), and with another which expresses PRG with acoding sequence interposed between it and the promoter (left, dottedline).

FIG. 19 depicts four plasmids designed to express retroviral proteins inmammalian cells. pSVgp and pRSVenv are cotransfected with a selectablemarker, while pSVgp-DHFR and pRSVenv-phleo are the equivalent plasmidswith the selectable marker placed downstream of the viral protein-codingregions.

FIG. 20 depicts three sites of fusion of HIV env and MoMLV env aftersite-directed mutagenesis. The joint at the extracellular margin of thetransmembrane region is designated as A, while B and C indicatelocations of joints at the middle of the transmembrane region andcytoplasmic margin, respectively. The numbering is according tonucleotide numbers (RNA Tumor Viruses, Vol. II, Cold Spring Harbor,1985). ST, SR, SE are the starts of tat, rev and env while TT, TR, andTE are the corresponding termination sites.

FIGS. 21A and B depicts the substitution of U3 in a 5' LTR by aheterologous promoter/enhancer which can be fused to either the Sac I,Bssh II or other site in the region.

FIG. 22 illustrates a representative method for crossing transgenic miceexpressing viral protein or vector RNA.

DETAILED DESCRIPTION OF THE INVENTION

I. Immunostimulation

The ability to recognize and defend against foreign pathogens is centralto the function of the immune system. This system, through immunerecognition, must be capable of distinguishing "self" from "nonself"(foreign), which is essential to ensure that defensive mechanisms aredirected toward invading entities rather than against host tissues. Thefundamental features of the immune system are the presence of highlypolymorphic cell surface recognition structures (receptors) and effectormechanisms (antibodies and cytolytic cells) for the destruction ofinvading pathogens.

Cytolytic T lymphocytes (CTL) are normally induced by the display ofprocessed pathogen-specific peptides in conjunction with the MHC class Ior class II cell surface proteins. Also stimulated by this type ofantigen presentation are the generation and production antibodies,helper cells and memory cells. Within one embodiment of the presentinvention, presentation of immunogenic viral determinants in the contextof appropriate MHC molecules efficiently induces optimal CTL responseswithout exposing the patient to the pathogen. This vector approach toimmunostimulation provides a more effective means of inducing potentclass I-restricted protective and therapeutic CTL responses, because thetype of immunity induced by the vector more closely resembles thatinduced by exposure to natural infection. Based on current knowledge ofseveral viral systems, it is unlikely that exogenously supplied,nonreplicating viral antigens, such as peptides and purified recombinantproteins, will provide sufficient stimulus to induce optimal classI-restricted CTL responses. Alternatively, vector-delivered expressionof selected viral proteins or other antigens corresponding to apathogenic condition, such as cancer, within target cells as describedwithin the present invention provides such a stimulus.

By way of example, in the case of HIV-1 infections, patients developantibodies specific for a variety of viral envelope-region determinants,some of which are capable of in vitro virus neutralization.Nevertheless, disease progression continues and the patients eventuallysuccumb to the disease. Low-level CTL responses against infectedpatients' cells (Plata et al., Nature 328:348-351, 1987) and againsttarget cells infected with recombinant vaccinia vectors expressing HIVgag, pol, or env (Walker et al., Nature 328:345-348, 1987; Walker etal., Science 240:64-66, 1988) have been detected in some HIV-1seropositive patients. In addition, it has recently been shown thatmurine as well as human CTL can be induced by autologous stimulatorcells expressing HIV gp 120 via transfection (Langlade-Demoyan et al.,J. Immunol. 141:1949, 1988). Improved CTL induction could betherapeutically advantageous to infected patients and provide effectivepreventive therapy to individuals under noninfectious conditions. HIVinfection itself may not be producing an adequate CTL response becauseother elements associated with HIV infection may prevent proper immunestimulation. In addition, it may be that stimulation of T-cells byinfected cells is an interaction that leads to infection of thestimulated T-cells.

HIV is only one example. This approach should be effective against manyvirally linked diseases or cancers where a characteristic antigen (whichdoes not need to be a membrane protein) is expressed, such as in humanpapillomavirus (HPV) and cervical carcinoma, HTLV-I-induced leukemias,prostate-specific antigen (PSA) and prostate cancer, mutated p53 andcolon carcinoma, GD2 antigen and melanoma. Example 1 describesprocedures for constructing plasmids capable of generating retroviralvectors in packaging cells, which then lead to expression of HIV viralantigens.

EXAMPLE 1 Vectors Expressing HIV Antigens

A. Env Expression Vector (See FIG. 1):

A 2.7 kb Kpn-Xho I DNA fragment was isolated from the HIV proviral cloneBH10-R3 (for sequence, see Ratner et al., Nature 313:277, 1985) and a≈400 bp Sal-Kpn I DNA fragment from IIIexE7deltaenv (a Bal31 deletion tont. 5496) was ligated into the Sal I site in the plasmid SK⁺. From thisclone, a 3.1 kb env DNA fragment (Xho I-Cla I) which also encodes rev,essential for env expression, was purified and ligated into a retroviralvector called pAFVXM (see Kriegler et al., Cell 38:483, 1984). Thisvector was modified in that the Bgl II site was changed by linkerinsertion to a Xho I site to facilitate cloning of the HIV env codingDNA fragment.

A dominant selectable marker gene comprised of a SV40 early promoterdriving expression of neomycin phosphotransferase gene was inserted intothe vector at the Cla I site to facilitate isolation of infected andtransfected cell lines. This vector is called pAF/Env^(r) /SV₂ neo (seeFIG. 1).

The Xho I site upstream from the ENV gene in the vector provides aconvenient site to insert additional promoters into the vector constructas the RSV promoter, SV40 early or late promoter, the CMV immediateearly (IE) promoter, human beta-actin promoter, and Moloney murine MLVSL3-3 promoter.

One such promoter, the CMV Immediate Early gene promoter (see FIG. 1), a673 bp DNA fragment Hinc II to Eag I, results in a tenfold increase inENV expression in a human T-cell line called Sup T1 when compared to theparental construct pAF/Env^(r) /SV₂ neo (see FIG. 2).

To improve titres of the vector one can use a recombinant retrovirusbased on N2 (Armentano et al., J. Virol. 61:1647-1650, 1987; Eglitas etal., Science 230:1395-1398, 1985). This vector contains both thepackaging sequences from N2 as well as the bacterial neomycinphosphotransferase gene. The above HIV env construct linked to the CMVpromoter was inserted into the unique Xho I site in N2.

B. Gag Expression Vector:

To efficiently express HIV gag and pol gene products in a retrovirusvector, two criteria must be met: 1) a REV response element must beadded to the 5 vector to override repressive elements buried in gag andpol; and 2) REV must be efficiently expressed to interact with theREV-responsive element inserted in the vector, thus allowing for correcttransport of viral messenger RNA into the cytoplasm.

A 2.5 kb Sac I-Eco RV DNA fragment was isolated from pBH10-R3 (seeRatner et al., op. cit.) and ligated into the Sac I-Sma I site of pUC31along with a linker coding for a universal translation terminationcodon. pUC31 is derived from pUC19 with additional Xho I, Bgl II, Bst IIand Nco I sites inserted between the Eco R1 and Kpn I sites of the polylinker. However, this construct contained the major splice donor (SD)site from HIV and thus could be problematic in virus generation. The SDsite was removed by subcloning a 70 bp Rsa I-Cla I fragment with a 2.1kb Cla I-Bam HI DNA fragment into the Hinc II-Bam HI site of SK⁺. TheBain HI site was converted into a Cla I site by linker insertion. Thisconstruct was designated SK⁺ gag protease SD delta.

A gag/pol SD deletion complete construct was produced by a three-partligation reaction in which a 757 bp Xho-Spe I fragment from SK⁺ gagprotease SD delta and a 4.3 kb Spe I-Nco I fragment from BH10 R3 wereinserted into SK⁺ XhoI-Nco I. The Xba I site in SK⁺ was converted to aNco I to facilitate this reaction.

In order to introduce both REV and the REV responsive elements in thevector, a 1.4 kb Ssp I deletion in the plasmid SK⁺ HIV env wasgenerated. This deletion removed intronic sequences which are notimportant for REV expression (REV expression will continue to be from aspliced mRNA.) In addition, this deletion does not effect the REVresponsive element located in env. The 1.1 kb DNA fragment coding forthe dominant selectable marker Neo, engineered to contain a eukaryotictranslation initiation codon, was introduced into the construct at theBgl II site in env. Insertion of neo facilitates detection of passagedvirus as well as selection for virus in an unspliced state duringpassage. A promoter such as the CMV is inserted into the XhoI site ofthis construct. This construct is designated SK⁺ CMV/REV/Neo. The finalviral construct may be produced by a four-part ligation reaction. A 2.5kb Xho I-Xba I DNA fragment from SK⁺ gag polymerase SD delta, a 3.5 kbSpe I-Cla I DNA fragment from SK⁺ CMV/REV/Neo and a 1.2 kb Cla I-HindIII DNA fragment from N2R3(-) (a subclone of N2 containing only the 3'LTR) are inserted into pUC N2R5 (a subclone of N₂ containing the 5' LTR)at the Xho I-Hind III site of this construct.

These plasmids, when placed in a suitable packaging cell, expressed aretroviral vector construct which contains a packaging signal. Thepackaging signal directed packaging of the vector construct into acapsid and envelope along with all further proteins required for viableretroviral particles. The capsid, envelope, and other proteins arepreferably produced from one or more plasmids containing suitablegenomes placed in the packaging cell. Such genomes may be proviralconstructs, which in a simple case may merely have the packaging signaldeleted. As a result, only the vector will be packaged. Suitablepackaging or packaging cell lines, and the genome necessary foraccomplishing such packaging, are described in Miller et al. (Mol. Cell.Bio. 6:2895, 1986), which is incorporated herein by reference. Asdescribed by Miller et al., it is preferable that further changes bemade to the proviral construct other than simple deletion of thepackaging signal in order to reduce the chances of recombination eventsoccurring within the packaging cell line, which may result in productionof viral particles which are not replication defective.

It will be understood that Example 1 is merely illustrative of aprocedure for generating an HIV envelope glycoprotein (gp) or otherviral antigen. It is also possible to provide a proviral vectorconstruct which expresses a modified HIV envelope gp on the target cellswhich will likewise stimulate an immune response, but with less T-cellcytopathic effects. Envelope glycoproteins can be suitably modifiedusing techniques well known in the art, for instance through use of thedisclosure of articles such as Kowalski et al. (Science 237:1351, 1987),which is herein incorporated by reference. Thus, a proviral constructmay be constructed by the above technique which generates retroviralconstructs expressing such a suitably modified gp. This construct isthen placed in a packaging cell as described above. The resultingrecombinant retroviruses produced from the packaging cell lines may beused in vitro and in vivo to stimulate an immune response through theinfection of susceptible target cells. It will be appreciated that otherproteins expressed from the HIV genome, such as gag, pol, vif, nef,etc., may also elicit beneficial cellular responses in HIV-infectedindividuals. Proviral vectors such as those described below are designedto express such proteins so as to encourage a clinically beneficialimmune response. It may be necessary for certain vectors to include revcoding sequences as well as a rev responsive element (Rosen et al.,Proc. Natl. Acad. Sci. 85:2071, 1988).

The following example demonstrates the ability of this type of treatmentto elicit CTL responses in mice.

EXAMPLE 2 Immune Response to Retroviral Vector-Encoded Antigens

A murine tumor cell line (B/C10ME) (H-2^(d)) was infected with arecombinant retrovirus carrying the pAF/Env^(r) /SV₂ neo vectorconstruct coding for HIV env.

One cloned HIV-env expressing cell line (B/C10ME-29) was then utilizedto stimulate HIV-env-specific CTL in syngeneic (i.e., MHC identical)Balb/c (H-2^(d)) mice (see FIG. 3). Mice were immunized byintraperitoneal injection with B/C10ME-29 cells (1×107 cells) andboosted on day 7-14. Responder spleen cell suspensions were preparedfrom these immunized mice and the cells cultured in vitro for 4 days inthe presence of either B/C10ME-29 (BCenv) or B/C10ME (BC)mitomycin-C-treated cells at a stimulator:responder cell ratio of 1:50(FIG. 3). The effector cells were harvested from these cultures,counted, and mixed with radiolabeled (⁵¹ Cr) target cells (i.e.,B/C10MEenv-29 or B/C10ME) at various effector:target (E:T) cell ratiosin a standard 4-5 hr ⁵¹ Cr-release assay. Following incubation, themicrotiter plates were centrifuged, 100 ul of culture supernate wasremoved, and the amount of radiolabel released from lysed cellsquantitated in a Beckman gamma spectrometer. Target cell lysis wascalculated as: % Target Lysis=Exp CPM-SR CPM/MR CPM-SR CPM×100, whereexperimental counts per minute (Exp CPM) represents effectors plustargets; spontaneous release (SR) CPM represents targets alone; andmaximum release (MR) CPM represents targets in the presence of 1M HCl.

The results (FIG. 4A) illustrate that CTL effectors were induced whichspecifically lysed HIV-env-expressing target cells (Bcenv) significantlymore efficiently than non-HIV env BC targets. Primed spleen cellsrestimulated in vitro with non-HIV-env-expressing control cells(B/C10ME) did not show significant CTL activity on either B/C10MEenv-29or B/C10ME targets, particularly at lower E:T cell ratios. Spleen cellsobtained from naive nonimmunized Balb/c mice which were stimulated invitro with B/C10MEenv-29 did not generate CTL (data not shown), thussuggesting the importance of the in vivo priming and boosting event.This experiment has been repeated and similar results obtained.

In another experiment, effector cells obtained from Balb/c miceimmunized, boosted and restimulated in vitro with a different H-2^(d)HIV-env-expressing tumor cell clone (L33-41) infected with the samepAF/Env^(r) /SV₂ neo (HIV-env) vector construct were capable of lysingB/C10MEenv-29 target cells. This provides additional support that theCTL generated in these mice are specifically recognizing an expressedform of HIV-env rather than simply a unique tumor cell antigen on thesecells. This result also suggests that the vector-delivered antigen ispresented in a similar manner by the two tumor cell lines. Thespecificity of the CTL response was further demonstrated by testingeffector cells obtained from BCenv immunized mice on BCenv target cellsexpressing the neo and HIV env genes, BC (non-neo, non-HIV env) parentaltargets and BCneo target cells expressing the neo resistance markergene, but no HIV env. FIG. 4B indicates that the CTL responses arespecific for the HIV env protein.

In another experiment, effector cells obtained from mice immunized with1×107 BCenv cells, boosted and restimulated in vitro, were treated withT-cell-specific monoclonal antibodies (Mab) plus complement (C') inorder to determine the phenotype of the induced cytotoxic effectorcells. Effectors were treated with either anti-Thy 1.2 (CD3), anti-L3T4(CD4) or anti-Lyt 2.2 (CD8) Mab for 30 minutes at 4° C., washed 1 timein Hank's balanced salt solution (HBSS), resuspended in low tox rabbitC' and incubated 30 minutes at 37° C. The treated cells were washed 3times in RPMI 1640 complete medium, counted, and tested for theirability to lyse BCenv radio-labeled target cells as previouslydescribed. FIG. 4C shows that treatment with either anti-Thy 1.2 oranti-Lyt 2.2 Mab+C' abrogated cytotoxic activity, whereas treatment withanti-L3T4 Mab +C' or C' alone did not significantly affect cytotoxicity.These results indicate that the majority population of cytotoxiceffector cells generated in this system are of the CD3⁺ CD4⁻ CD8⁺cytotoxic T-cell phenotype.

Experiments were performed to determine the mHC restriction of CTLeffector cells described above. Polyclonal antibodies directed againstdifferent H-2 regions of the murine MHC (i.e., anti-H-2^(d),anti-H-2D^(d), anti-H-2L^(d), anti-H-2K^(d), anti-H-2I^(d)) were used toinhibit the CTL response on BCenv target cells. The anti-H-2^(k)antiserum was used as a negative control. The data (FIG. 4D) indicatethat the Balb/c anti-BCenv CTL response is inhibited primarily by theanti-H-2D^(d) antiserum. This suggests that these CTL responses arerestricted by MHC class I molecules, most likely encoded within the Dregion of the H-2 complex.

In addition to experiments in which mice were immunized withreplication-competent HIV env-expressing tumor cells, tests wereconducted to determine whether proliferating stimulator cells werenecessary for inducing CTL in vivo. Mice were immunized with eitherirradiated (10,000 rads) or nonirradiated BCenv cells, and the primedspleen cells were later stimulated in vitro, as previously described.The resulting effector cells were tested for CTL activity onradio-labeled BCenv and BC target cells. FIG. 4E indicates HIV-specificCTL can be induced in vivo with either irradiated or nonirradiatedstimulator cells. These data demonstrate that CTL induction by HIVenv-expressing stimulator cells is not dependent upon proliferation ofstimulator cells in vivo and that the presentation of HIV env antigen inthe appropriate MHC context is sufficient for effective CTL induction.

Formalin fixed cells also elicit an equivalent immune response. Thisshows that killed cells or perhaps cell membranes expressing theappropriate antigen in the proper MHC class I/II molecular context aresufficient for induction of effective CTL responses.

Additional experiments were performed to examine the optimal injectiondose of BCenv cells into Balb/c mice. Mice were immunized with varyingnumbers of BCenv stimulator cells, restimulated in vitro as described,and tested for CTL activity. The results shown in FIG. 4F indicate thatimmunization of mice with 5×10⁶ env-expressing BCenv-29 stimulator cellsgenerated an optimal CTL response under these conditions.

Further experiments examined the ability of vector-infected HIVenv-expressing BCenv stimulator cells to induce CTL responses in otherH-2^(d) mouse strains other than Balb/c, in order to provide anindication as to genetic restrictions imposed on host responsiveness.Different strains of H-2^(d) (i.e., Balb/c, DBA/2, B10.D2), as well asH-2^(d) ×H-2^(b) F1 hybrid mice i.e., CB6F1 (Balb/c×B6 F1); B6D2F1(B6×DBA/2 F1)!, were immunized with BCenv stimulator cells and examinedfor the induction of CTL responses. FIG. 4G illustrates that all strainsincluding F1 hybrids generate CTL responses against the BCenv targetcells to varying degrees. Although some strains also exhibit responsesagainst the parental (i.e., non-HIV env) target cells, these responsesare lower than those directed against the BCenv target.

Implementation of this immunostimulant application in humans requiresthat (1) the gene coding for the antigen of interest be delivered tocells, (2) the antigen be expressed in appropriate cells, and (3) MHCrestriction requirements, i.e., class I and class II antigeninteraction, are satisfied. Within a preferred embodiment, preparationsof vector are made by growing the producer cells in normal medium,washing the cells with PBS plus human serum albumin (HSA) at 10 mg/ml,then growing the cells for 8-16 hours in PBS plus HSA. Titres obtainedare typically 10⁴ to 10⁶ /ml depending on the vector, packaging line orparticular producer line clone. The vector supernatants are filtered toremove cells and are concentrated up to 100-fold by filtration through100,000 or 300,000 pass Amicon filters (Wolff et al., Proc. Natl. Acad.Sci. 84:3344, 1987). This lets globular proteins of 100,000 or 300,000pass but retains 99% of the viral vector as infectious particles. Thestocks can be frozen for storage since they lose about 50% of theinfectious units on freezing and thawing. The most direct deliveryinvolves administration of the appropriate gene-carrying vector into theindividual and reliance upon the ability of the vector to efficientlytarget to the appropriate cells, which can then initiate stimulation ofthe immune response. The dose is generally 10⁵ to 10⁶ infectiousunits/kg body weight. However, a more practical approach may involve theextracorporeal treatment of patient peripheral blood lymphocytes (PBL),fibroblasts or other cells obtained from each individual with thevector. PBL can be maintained in culture through the use of mitogens(phytohemagglutinin) or lymphokines (e.g., IL-2). This type of approachallows for directed vector infection, monitoring of expression andexpansion of the antigen presenting cell population prior to injection,and return of vector-expressing cells to the respective patient. Othertypes of cells can also be explanted, vector introduced, and the cellsreturned to the patient. Only a moderate number of infected cells (10⁵-10⁷) is necessary to elicit strong immune responses in mice. It isprobable that the dose to elicit an immune response is roughly the sameper individual animal or patient with very little dependence on bodysize.

Within one alternative method, cells are infected ex vivo as describedabove, and either inactivated by irradiation (see FIG. 4E) or killed byfixation, such as by formalin. Formalin fixation of cells treated with avector expressing HIV env after treatment with the vector carrying theHIV env gene induces a strong CTL response.

Within another alternative method, stimulator cell membrane fragmentswhich contain both the antigen of interest and the appropriate MHCmolecule as a complex are employed. Cells are infected with vector,genes expressed, cells disrupted and the membranes purified bycentrifugation or affinity columns specific for the MHC-antigen complex.This process provides greater quality control from a manufacturing andstability standpoint.

This approach also allows the use of cells that normally do not expresshuman MHC molecules (e.g., human cell mutants, mouse cells). IndividualMHC class I or class II genes are infected into MHC-cells to giveexpression of the individual corresponding MHC protein, in a particularcell line. Thus, a bank of cell lines capable of displaying antigens inthe context of different MHC classes is generated. A small number ofthese (10-20) will cover (i.e., have a match with) the majority of thehuman population. For example, HLA A2 is present in about 30-60% ofindividuals. In the case of non-human cells, those can be derived fromtransgenic animals (such as mice) which express human MHC moleculesgenerally or in specific tissues due to the presence of a transgene inthe strain of animals (see, e.g., Chamberlin et al., Proc. Natl. Acad.Sci. USA 85: 7690-7694, 1988).

In any of the above situations, the presentation or response to thepresentation can be enhanced by also infecting into the cells genes ofother proteins involved in the immune interactions which are missing orunderrepresented (e.g., β microglobulin, LFA3, CD3, ICAM-I and others).β microglobulin is a nonvariant, necessary subunit of the class I MHC,CD3 is involved in the MHC interaction, and LFA3 and ICAM-I moleculesenhance the interaction of cells of the immune system (see, e.g.,Altmann et al., Nature 338:512, 1989) leading to stronger responses tothe same level of immune stimulation.

In the case of transgenic mice expressing human MHc, the stimulationcould also be performed in the mouse using somatic transgenic mousecells expressing a foreign antigen, the gene for which was introduced bya viral vector or other means, as stimulators. The mouse CTL thusgenerated would have T-cell receptors expressing in the context of thehuman MHC, and could be used for passive cellular immunization ortreatment (i.e., infused into patients) of patients.

As a further alternative, one can use cells from a patient and boostexpression of "self" MHC class I genes by introducing the matched MHCgene by vector transfer or other means. Such a boost in MHC I expressioncauses more efficient presentation of foreign antigens, whether they arepresent already in the patient's cells (e.g., tumor cells) orsubsequently added using viral vectors encoding foreign antigens. This,in turn, leads to a more potent immune response when even cells withreduced MHC I expression (such as some virally infected cells or sometumor types) are efficiently eliminated. Within certain aspects of thepresent invention, one can infect susceptible target cells with acombination or permutation of nucleic acid sequences encoding (a)individual Class I or Class II MHC protein, or combinations thereof; (b)specific antigens or modified forms thereof capable of stimulating animmune response; and (c) other proteins involved in the immuneinteractions which are missing or underrepresented, as discussed above.The respective steps of infection may be performed in vivo or ex vivo.

A different form of administration is the implantation of producer linesmaking retroviral vector particles. These may be immunologicallyunmatched classical producer cell lines or the patients own cells, whichhave been explanted, treated and returned (see VI Alternative ViralVector Packaging Techniques, below). Both types of implants (10⁵ -10⁶cells/kg body weight) would have a limited life span in the patient, butwould lead to the retroviral vector infecting large numbers (10⁷ -10¹⁰)of cells in their vicinity in the body.

In any case, the success of the HIV immune stimulating treatment can beassayed by removing a small amount of blood and measuring the CTLresponse using as targets the individual's own cells infected withvector leading to env expression.

When it is desired to stimulate an MHC class I or class II restrictedimmune response to pathogens, including pathogenic viruses other thanHIV, suitable forms of envelope or other antigens associated with suchretroviruses which will stimulate an immune response can be ascertainedby those skilled in the art. In general, there will be combinations ofepitopes which cause induction of various parts of the immune system(e.g., T_(H) -, T_(C) -, B-cells). In addition, some epitopes may bepathogenic or hypervariable but immunodominant. The present inventionallows a "mix-and-match" selection of combinations of desirable epitopesand exclusion of undesirable epitopes. For example, in HIV, a number ofhypervariable loops which carry immunodominant B- and T-cell epitopescan be strung together in the gene sequence carried by the vector sothat the resultant immunostimulation is appropriate for thepreponderance of HIV strains found clinically.

An alternative approach to creating a desired immune response is todeliver an antigen-specific T-cell receptor gene to an appropriate cell,such as a T-cell. It is also possible to molecularly graft the geneticmessage for antigen recognition sites of immunoglobulin molecules intothe corresponding sites in the genes of the related T-cell receptorsubunits α and β. Such altered protein molecules will not be MHCrestricted, and will be able to perform as T_(H) - and T_(C) -cellsspecific for the antigen defined by the original immunoglobulin. Anothertactic is to transfer genes for effector molecules in NK into NK cellsto confer additional non-MHC limited killing capability on these cells.In addition, specific immunoglobulin genes could similarly be usefulwhen delivered to B-cells to cause the large-scale in vivo production ofa particular antibody molecule in a patient.

II. Blocking Agents

Many infectious diseases, cancers, autoimmune diseases, and otherdiseases involve the interaction of viral particles with cells, cellswith cells, or cells with factors. In viral infections, viruses commonlyenter cells via receptors on the surface of susceptible cells. Incancers, cells may respond inappropriately or not at all to signals fromother cells or factors. In autoimmune disease, there is inappropriaterecognition of "self" markers. Within the present invention, suchinteractions may be blocked by producing, in vivo, an analogue to eitherof the partners in an interaction.

This blocking action may occur intracellularly, on the cell membrane, orextracellularly. The blocking action cf a viral or, in particular, aretroviral vector carrying a gene for a blocking agent, can be mediatedeither from inside a susceptible cell or by secreting a version of theblocking protein to locally block the pathogenic interaction.

In the case of HIV, the two agents of interaction are the gp 120/gp 41envelope protein and the CD4 receptor molecule. Thus, an appropriateblocker would be a vector construct expressing either an HIV envanalogue that blocks HIV entry without causing pathogenic effects, or aCD4 receptor analogue. The CD4 analogue would be secreted and wouldfunction to protect neighboring cells, while the gp 120/gp 41 issecreted or produced only intracellularly so as to protect only thevector-containing cell. It may be advantageous to add humanimmunoglobulin heavy chains or other components to CD4 in order toenhance stability or complement lysis. Delivery of a retroviral vectorencoding such a hybridsoluble CD4 to a host results in a continuoussupply of a stable hybrid molecule.

Vector particles leading to expression of HIV env may also beconstructed as described above. It will be evident to one skilled in theart which portions are capable of blocking virus adsorption withoutovert pathogenic side effects (Willey et al., J. Virol. 62:139, 1988;Fisher et al., Science 233:655, 1986). The following example describesthe construction of a CD4 vector from which infectious vector particleswere made (FIG. 5).

EXAMPLE 3 sCD4 Vector

1. A 1.7 kb Eco R1- Hind III DNA fragment from pMV7.T4 (Maddon et al.,Cell 47:333, 1986) was blunt-end ligated to the Hinc II site of Sk⁺.

2. A universal translation termination sequence containing an Xba I sitewas inserted into the Nhe I site of the CD4 fragment.

3. The 1.7 kb Xho I-Cla I fragment was excised and cloned into the XhoI-Cla I site of pAFVXM. These vector plasmids can be used to generateinfectious vector particles, as described in Example 1.

Such infectious blocking vectors, when put into human T-cell lines inculture, can inhibit the spread of HIV infections. Preparation,concentration and storage of infectious retroviral vector preparationsis as for the immunostimulant. Route of administration would also be thesame, with doses about tenfold higher. Another route which may be usedis the aspiration of bone marrow, infection with retroviral vector andreturn of this infected marrow (Gruber et al., Science 230:1057, 1985)to the patient. Since the marrow replication will amplify the vectorexpression through cell replication, doses in the range of theimmunostimulant can be used (10⁵ -10⁶ /kg body weight).

In any case, the efficacy of the treatment can be assayed by measuringthe usual indicators of disease progression, including antibody level,viral antigen production, infectious HIV levels, or levels ofnonspecific infections.

III. Expression of Palliatives

Techniques similar to those described above can be used to producerecombinant retroviruses with vector constructs which direct theexpression of an agent (or "palliative") which is capable of inhibitinga function of a pathogenic agent or gene. Within the present invention,"capable of inhibiting a function" means that the palliative eitherdirectly inhibits the function or indirectly does so, for example, byconverting an agent present in the cells from one which would notnormally inhibit a function of the pathogenic agent to one which does.Examples of such functions for viral diseases include adsorption,replication, gene expression, assembly, and exit of the virus frominfected cells. Examples of such functions for a cancerous cell orcancer-promoting growth factor include viability, cell replication,altered susceptibility to external signals (e.g., contact inhibition),and lack of production or production of mutated forms of anti-oncogeneproteins.

(i) Inhibitor Palliatives

In one aspect of the present invention, the recombinant retroviruscarries a vector construct which directs the expression of a gene whichcan interfere with a function of a pathogenic agent, for instance inviral or malignant diseases. Such expression may either be essentiallycontinuous or in response to the presence in the cell of another agentassociated either with the pathogenic condition or with a specific celltype (an "identifying agent"). In addition, vector delivery may becontrolled by targeting vector entry specifically to the desired celltype (for instance, a virally infected or malignant cell) as discussedbelow.

A preferred method of administration is leukophoresis, in which about20% of an individual's PBLs are removed at any one time and manipulatedin vitro. Thus, approximately 2×10⁹ cells may be treated and replaced.Since the current maximum titres are around 10⁶ /ml, this requires 2 to20 liters of starting viral supernatant. Repeat treatments also would beperformed. Alternatively, bone marrow may be treated and allowed toamplify the effect as described above.

In addition, packaging cell lines producing a vector may be directlyinjected into a subject, allowing continuous production of recombinantvirions. Examples of suitable cell types include monocytes, neutrophils,or their progenitors, since these cells are present in the peripheralblood but can also leave the circulatory system to allow virusproduction in extravascular tissue (particularly the central nervoussystem) where virion production may be therapeutically required. Such acell line would ultimately be rejected as foreign by the host immunesystem. To ensure the eventual destruction of these foreign cells fromthe host (even an immuno-suppressed host) the cell line may beengineered to express the gene for a conditionally lethal protein, suchas HSVTK. Thus, administration of the drug Acyclovir (ACV) (a drug whichis specifically toxic for cells expressing HSVTK) eliminates these cellsafter sufficient vector has been produced in vivo. Such a packaging cellline could be a continuous cell line or could be made directly from hostcells.

In one embodiment, retroviral viruses which express RNA complementary tokey pathogenic gene transcripts (for example, a viral gene product or anactivated cellular oncogene) can be used to inhibit translation of thattranscript into protein, such as the inhibition of translation of theHIV tat protein. Since expression of this protein is essential for viralreplication, cells containing the vector would be resistant to HIVreplication. To test this, the vector atat (FIG. 10) has beenconstructed, packaged as recombinant virions and introduced into humanT-cells and monocyte cell lines in the absence of replication-competenthelper virus.

In a second embodiment, where the pathogenic agent is a single-strandedvirus having a packaging signal, RNA complementary to the viralpackaging signal (e.g., an HIV packaging signal when the palliative isdirected against HIV) is expressed, so that the association of thesemolecules with the viral packaging signal will, in the case ofretroviruses, inhibit stem loop formation or tRNA primer bindingrequired for proper encapsidation or replication of the retroviral RNAgenome.

In a third embodiment, a retroviral vector may be introduced whichexpresses a palliative capable of selectively inhibiting the expressionof a pathogenic gene, or a palliative capable of inhibiting the activityof a protein produced by the pathogenic agent. In the case of HIV, oneexample is a mutant tat protein which lacks the ability to transactivateexpression from the HIV LTR and interferes (in a transdominant manner)with the normal functioning of tat protein. Such a mutant has beenidentified for HTLV II tat protein ("IXII Leu⁵ " mutant; see Wachsman etal., Science 235:674, 1987). A mutant transrepressor tat should inhibitreplication much as has been shown for an analogous mutant repressor inHSV-1 (Friedmann et al., Nature 335:452, 1988).

Such a transcriptional repressor protein may be selected for in tissueculture using any viral-specific transcriptional promoter whoseexpression is stimulated by a virus-specific transactivating protein (asdescribed above). In the specific case of HIV, a cell line expressingHIV tat protein and the HSVTK gene driven by the HIV promoter will diein the presence of ACV. However, if a series of mutated tat genes areintroduced to the system, a mutant with the appropriate properties(i.e., represses transcription from the HIV promoter in the presence ofwild-type tat) will grow and be selected. The mutant gene can then bereisolated from these cells. A cell line containing multiple copies ofthe conditionally lethal vector/tat system may be used to assure thatsurviving cell clones are not caused by endogenous mutations in thesegenes. A battery of randomly mutagenized tat genes are then introducedinto these cells using a "rescuable" retroviral vector (i.e., one thatexpresses the mutant tat protein and contains a bacterial origin ofreplication and drug resistance marker for growth and selection inbacteria). This allows a large number of random mutations to beevaluated and permits facile subsequent molecular cloning of the desiredmutant cell line. This procedure may be used to identify and utilizemutations in a variety of viral transcriptional activator/viral promotersystems for potential antiviral therapies.

In a fourth embodiment, the recombinant retrovirus carries a vectorconstruct that directs the expression of a gene product capable ofactivating an otherwise inactive precursor into an active inhibitor ofthe pathogenic agent. For example, the HSVTK gene product may be used tomore effectively metabolize potentially antiviral nucleoside analogues,such as AZT or ddC. The HSVTK gene may be expressed under the control ofa constitutive macrophage or T-cell-specific promoter and introducedinto these cell types. AZT (and other nucleoside antivirals) must bemetabolized by cellular mechanisms to the nucleotide triphosphate formin order to specifically inhibit retroviral reverse transcriptase andthus HIV replication (Furmam et al., Proc. Natl. Acad. Sci. USA83:8333-8337, 1986). Constitutive expression of HSVTK (a nucleoside andnucleoside kinase with very broad substrate specificity) results in moreeffective metabolism of these drugs to their biologically activemucleotide triphosphate form. AZT or ddC therapy will thereby be moreeffective, allowing lower doses, less generalized toxicity, and higherpotency against productive infection. Additional nucleoside analogueswhose nucleotide triphosphate forms show selectivity for retroviralreverse transcriptase but, as a result of the substrate specificity ofcellular nucleoside and nucleotide kinases are not phosphorylated, willbe made more efficacious. A description of a representative method isset forth in Example 4.

EXAMPLE 4 Vectors Designed to Potentiate the Antiviral Effect of AZT andAnalogues

A. All of the following retroviral vectors are based on the "N2" vector(see Keller et al., Nature 318:149-154, 1985). Consequently, 5' and 3'Eco R1 LTR fragments (2.8 and 1.0 kb, respectively) were initiallysubcloned into plasmids containing polylinkers (into SK⁺ to give pN2R5+/-!; into pUC31 to give p31N2R5 +/-! and p31N2R3 +/-! to facilitatevector construction. pUC31 is a modification of pUC19 carryingadditional restriction sites (Xho I, Bgl II, BssH II, and Nco I) betweenthe Eco R1 and Sac I sites of the polylinker. In one case, a 1.2 kb ClaI/Eco R1 5' LTR fragment was subcloned into the same sites of an SK⁺vector to give pN2CR5. In another case, the 5' LTR containing a 6 bpdeletion of the splice donor sequence was subcloned as a 1.8 kb Eco R1fragment into pUC31 (p31N25delta +!). The coding region andtranscriptional termination signals of HSV-1 thymidine kinase gene wereisolated as a 1.8 kb Bgl II/Pvu II fragment from plasmid 322TK (3.5 kbBam HI fragment of HSVTK cloned into Bam HI of pBR322) and cloned intoBgl II/Sma I-digested pUC31 (pUCTK). For constructs which requiredeletion of the terminator signals, pUCTK was digested with Sma I andBam HI. The remaining coding sequences and sticky-end Bam HI overhangwere reconstituted with a double-stranded oligonucleotide made from thefollowing oligomers:

5' GAG AGA TGG GGG AGG CTA ACT GAG 3' and 5' GAT CCT CAG TTA GCC TCC CCCATC TCT C 3' forming the construct pTK delta A.

For diagnostic purposes, the oligos were designed to destroy the Sma Isite while keeping its Ava I site without changing the translatedprotein.

The 0.6 kb HIV promoter sequences were cloned as a Dra I/Hind IIIfragment from pCV-1 (see Arya et al., Science 229:69-73, 1985) into HincII/Hind III-cut SK⁺ (SKHL).

B. Construction of TK-1 and TK-3 retroviral vectors (see FIG. 6).

1. The 5 kb Xho I/Hind III 5' LTR and plasmid sequences were isolatedfrom p31N2R5(+).

2. HSVTK coding sequences lacking transcriptional termination sequenceswere isolated as a 1.2 kb Xho I/Bam HI fragment from pTKdeltaA.

3. 3' LTR sequences were isolated as a 1.0 kb Bam HI/Hind III fragmentfrom pN2R3(-).

4. The fragments from steps 1-3 were mixed, ligated, transformed intobacteria, and individual clones identified by restriction enzymeanalysis (TK-1).

5. TK-3 was constructed by linearizing TK-1 with Bam HI, filling in the5' overhang and blunt-end ligating a 5'-filled Cla I fragment containingthe bacterial lac UV5 promoter, SV40 early promoter, plus Tn5 Neo^(r)gene. Kanamycin-resistant clones were isolated and individual cloneswere screened for the proper orientation by restriction enzyme analysis.

These constructs were used to generate infectious recombinant vectorparticles in conjunction with a packaging cell line, such as PA317, asdescribed above.

Administration of these retroviral vectors to human T-cell andmacrophage/monocyte cell lines can increase their resistance to HIV inthe presence of AZT and ddC compared to the same cells withoutretroviral vector treatment. Treatment with AZT would be at lower thannormal levels to avoid toxic side effects, but still efficiently inhibitthe spread of HIV. The course of treatment would be as described for theblocker.

Preparation, concentration and storage of the retroviral vectorpreparations would be as described above. Treatment would be aspreviously described but ex corpore treatment of patients' cells wouldaim for uninfected potentially susceptible T-cells or monocytes. Onepreferred method of targeting the susceptible cell is with vectors whichcarry HIV env or hybrid env (see Section VIII Cell Line SpecificRetroviruses, below) to direct absorption of vector particles to CD4⁺cells. Normal adults have about 5×10⁹ T4 cells in their total blood andabout the same number of monocytes.

A fifth embodiment for producing inhibitor palliatives involves thedelivery and expression of defective interfering viral structuralproteins, which inhibit viral assembly. Vectors would code for defectivegag, pol, env or other viral particle proteins or peptides, and thesewould inhibit in a dominant fashion the assembly of viral particles.This occurs because the interaction of normal subunits of the viralparticle is disturbed by interaction with the defective subunits.

A sixth such embodiment involves the expression of inhibiting peptidesor proteins specific for viral protease. Viral protease cleaves theviral gag and gag/pol proteins into a number of smaller peptides.Failure of this cleavage in all cases leads to complete inhibition ofproduction of infectious retroviral particles. The HIV protease is knownto be an aspartyl protease, and these are known to be inhibited bypeptides made from amino acids from protein or analogues. Vectors toinhibit HIV will express one or multiple fused copies of such peptideinhibitors.

A seventh embodiment involves the delivery of suppressor genes which,when deleted, mutated or not expressed in a cell type, lead totumorigenesis in that cell type. Reintroduction of the deleted gene bymeans of a viral vector leads to regression of the tumor phenotype inthese cells. Examples of such cancers are retinoblastoma and WilmsTumor. Since malignancy can be considered to be an inhibition ofcellular terminal differentiation compared with cell growth, theretroviral delivery and expression of gene products which lead todifferentiation of a tumor should also, in general, lead to regression.

In an eighth embodiment, the retroviral construct (with or without theexpression of a palliative) provides a therapeutic effect by insertingitself into a virus, oncogene, or pathogenic gene, thereby inhibiting afunction required for pathogenesis. This embodiment requires thedirection of retroviral integration to a specific site in the genome byhomologous recombination, integrase modification, or other methods(described below).

In an ninth embodiment, the retroviral vector provides a therapeuticeffect by encoding a ribozyme (an RNA enzyme) (Haseloff and Gerlach,Nature 334:585, 1989) which will cleave and hence inactivate RNAmolecules corresponding to a pathogenic function. Since ribozymesfunction by recognizing a specific sequence in the target RNA and thissequence is normally 12 to 17 bp, this allows specific recognition of aparticular RNA species such as a RNA or a retroviral genome. Additionalspecificity may be achieved in some cases by making this a conditionaltoxic palliative (see below).

One way of increasing the effectiveness of inhibitory palliatives is toexpress viral inhibitory genes in conjunction with the expression ofgenes which increase the probability of infection of the resistant cellby the virus in question. The result is a nonproductive "dead-end" eventwhich would compete for productive infection events. In the specificcase of HIV, vectors may be delivered which inhibit HIV replication (byexpressing anti-sense tat, etc., as described above) and alsooverexpress proteins required for infection, such as CD4. In this way, arelatively small number of vector-infected HIV-resistant cells act as a"sink" or "magnet" for multiple nonproductive fusion events with freevirus or virally infected cells.

(ii) Conditional Toxic Palliatives

Another approach for inhibiting a pathogenic agent is to express apalliative which is toxic for the cell expressing the pathogeniccondition. In this case, expression of the palliative from the proviralvector should be limited by the presence of an entity associated withthe pathogenic agent, such as an intracellular signal identifying thepathogenic state in order to avoid destruction of nonpathogenic cells.This cell-type specificity may also be conferred at the level ofinfection by targeting recombinant retrovirus carrying the vector tocells having or being susceptible to the pathogenic condition.

In one embodiment of this method, a recombinant retrovirus (preferably,but not necessarily, a recombinant MLV retrovirus) carries a vectorconstruct containing a cytotoxic gene (such as ricin) expressed from anevent-specific promoter, such as a cell cycle-dependent promoter (e.g.,human cellular thymidine kinase or transferrin receptor promoters),which will be transcriptionally active only in rapidly proliferatingcells, such as tumors. In this manner, rapidly replicating cells, whichcontain factors capable of activating transcription from thesepromoters, are preferentially destroyed by the cytotoxic agent producedby the proviral construct.

In a second embodiment, the gene producing the cytotoxic agent is undercontrol of a tissue-specific promoter, where the tissue specificitycorresponds to the origin of the tumor. Since the viral vectorpreferentially integrates into the genome of replicating cells (forexample, normal liver cells are not replicating, while those of ahepatocarcinoma are), these two levels of specificity (viralintegration/replication and tissue-specific transcriptional regulation)lead to preferential killing of tumor cells. Additionally,event-specific and tissue-specific promoter elements may be artificiallycombined such that the cytotoxic gene product is expressed only in celltypes satisfying both criteria (e.g., in the example above, combinedpromoter elements are functional only in rapidly dividing liver cells)Transcriptional control elements may also be amplified to increase thestringency of cell-type specificity.

These transcriptional promoter/enhancer elements need not necessarily bepresent as an internal promoter (lying between the viral LTRS) but maybe added to or replace the transcriptional control elements in the viralLTRs which are themselves transcriptional promoters, such thatcondition-specific transcriptional expression will occur directly fromthe modified viral LTR. In this case, either the condition for maximalexpression will need to be mimicked in retroviral packaging cell lines(e.g., by altering growth conditions, supplying necessarytransregulators of expression or using the appropriate cell line as aparent for a packaging line), or the LTR modification is limited to the3' LTR U3 region, to obtain maximal recombinant viral titres. In thelatter case, after one round of infection/integration, the 3' LTR U3 isnow also the 5' LTR U3, giving the desired tissue-specific expression.

In a third embodiment, the proviral vector construct is similarlyactivated but expresses a protein which is not itself cytotoxic, andwhich processes within the target cells a compound or a drug with littleor no cytotoxicity into one which is cytotoxic (a "conditionally lethal"gene product). Specifically, the proviral vector construct carries theherpes simplex virus thymidine kinase ("HSVTK") gene downstream andunder the transcriptional control of an HIV promoter (which is known tobe transcriptionally silent except when activated by HIV tat protein).Expression of the tat gene product in human cells infected with HIV andcarrying the proviral vector construct causes increased production ofHSVTK. The cells (either in vitro or in vivo) are then exposed to a drugsuch as acyclovir or its analogues (FIAU, FIAC, DHPG). These drugs areknown to be phosphorylated by HSVTK (but not by cellular thymidinekinase) to their corresponding active nucleotide triphosphate forms(see, for example, Schaeffer et al., Nature 272:583, 1978). Acyclovirand FIAU triphosphates inhibit cellular polymerases in general, leadingto the specific destruction of cells expressing HSVTK in transgenic mice(see Borrelli et al., Proc. Natl. Acad. Sci. USA 85:7572, 1988). Thosecells containing the recombinant vector and expressing HIV tat proteinare selectively killed by the presence of a specific dose of thesedrugs. In addition, an extra level of specificity is achieved byincluding in the vector the HIV rev protein, responsive CRS/CARsequences. In the presence of the CRS sequence gene expression issuppressed, except in the presence of the CAR sequences and the revprotein. Example 5 provides an illustration of this technique.

EXAMPLE 5 Vector to Conditionally Potentiate the Toxic Action of ACV orIts Analogues

Construction of Vectors

A. Construction of PKTVIHAX (see FIG. 7).

1. The 9.2 kb Asu II/Xho I fragment was isolated from vector pN2 DNA.

2. The 0.6 kb Xho I/Bam HI promoter fragment was isolated from plasmidpSKHL.

3. The 0.3 kb Bgl II/Acc I and 1.5 kb Acc I/Acc I fragment were purifiedfrom pUCTK.

4. The fragments from 1, 2, and 3 were ligated, transformed intobacteria, and appropriate Amp^(r) clones of the given structureidentified by restriction enzyme analysis.

B. Construction of pKTVIH-5 and pKTVIH5 Neo retroviral vectors (seeFioure 8).

1. The 4.5 kb 5' LTR and vector fragment was isolated as an Xho I/Bam HIfragment from vector p31N25delta(+) .

2. The 1.0 kb 3' LTR was isolated as an Apa I/Bam HI fragment frompN2R3(+) fragment.

3. The 0.6 kb HIV promoter element was isolated from pSKHL as an ApaI/Eco R1 fragment.

4. The HSVTK coding sequence and transcriptional termination sequenceswere isolated as 1.8 kb Eco R1/Sal I fragment from PUCTK.

5. The fragments from 1-4 were combined, ligated, transformed intobacteria, and clones of the given structure were identified byrestriction enzyme analysis (pKTVIH-5).

6. Plasmid pKTVIH5 Neo was constructed by linearizing pKTVIH5 with ClaI; mixing with a 1.8 kb Cla I fragment containing the bacterial lac UV5promoter, SV40 early promoter, and Tn5 Neo^(r) marker, ligating,transforming bacteria and selecting for kanamycin resistance. Cloneswith the insert in the indicated orientation were identified byrestriction analysis.

C. Construction of MHMTK Neo retroviral vector (see FIG. 9).

1. Construction of intermediate plasmid MHM-1 LTR.

a) Plasmid pN2CR5 was linearized by partial digestion with Fok I, the 5'overhang filled in with deoxynucleotide triphosphates using Klenow DNApolymerase, and Hind III linkers inserted. After transformation intobacteria, a clone with a Hind III linker inserted in the MLV LTR Fok Isite was identified by restriction enzyme analysis (pN2CR5FH).

b) Plasmid pN2CR5FH was linearized with Nhe I, the 5' overhang filled inwith Klenow polymerase digested with Hind III, and the 4.3 kb fragmentwith promoterless MLV sequences isolated.

c) 0.5 kb Eco RV/Hind III HIV promoter sequences were isolated frompSKHL.

d) b and c were mixed, ligated, used to transform bacteria, and thestructure of MHM-1 was confirmed by restriction enzyme analysis.

2. The 0.7 kb Eco RV/Bal I fragment isolated from MHM-1 was subclonedinto the Eco RV site of plasmid I30B (a modified IBI30 plasmidcontaining additional Bgl II, Bst II, Neo I and Nde I sites in thepolylinker). After transformation into bacteria, clones with theappropriate orientation were identified by restriction enzyme analysis(pMHMB).

3. Plasmid pMHMB was digested with Apa I and Xho I and gel purified.

4. MHM-1 was digested with Apa I/Bam HI and the 1.8 kb MHMLTR/leadersequence gel purified.

5. The 2.8 kb Bgl II/Sal I fragment containing the HSVTK coding regionupstream of the SV40 early promoter driving Neo^(r) taken from pTK-3(see FIG. 3).

6. 3-5 were mixed, ligated, used to transform bacteria, and appropriateclones were identified by restriction enzyme analysis.

This vector and similar vectors which contain inducible elements intheir LTR's result in an added safety feature. Briefly, since the LTR isinactive in the absence of HIV, insertional downstream activation ofundesirable host genes (such as proto-oncogenes) does not occur.However, tat expression in the packaging cell line allows facilemanipulation of the virion in tissue culture.

D. Construction of RRKTVIH retroviral vector (see FIG. 10)

1. The 9.2 kb Asu II/Xho I fragment was isolated from vector pN2 DNA.

2. The 0.6 kb Xho I/Eco R1 HIV promoter fragment was isolated fromplasmid pSKHL.

3. The HIV rev responsive HSVTK (RRTK) was constructed in the followingmanner:

a) The HSVTK gene was subcloned as a 1.8 kb HinC II/Pvu II fragment intothe Eco RV site of vector SK⁺ (pSTK -!).

b) The 1.8 kb Kpn I/Hind III fragment which contains the CRS/CARelements from HIV env was repaired and blunt-end ligated into the Sma Isite of vector I30B (pCRS/CAR +/-!). I30B is a modified IBI30 plasmidcontaining the same additional restriction sites as for pUC31 with anNde I site instead of the IBI30 Xho I site.

c) The 3.6 kb BssH II/Eco R1 fragment containing vector and HSVTKpolyadenylation signals was isolated from pSTK(-),

d) The 1.8 kb Bam HI/BssH II CRS/CAR fragment was isolated frompCRS/CAR(-).

e) The 1.2 Eco R1/Bam HI coding sequence fragment was isolated frompTKdeltaA.

f) C, D and E were ligated and appropriate recombinants screened byrestriction enzyme analysis.

4. Rev-responsive HSVTK was isolated as a 3.6 kb Eco R1/Cla I fragment.

5. 1, 2 and 4 were ligated and appropriate recombinants identified byrestriction enzyme analysis.

E. Construction of tat and anti-tat expression vectors (see FIG. 11).

These vectors are used as pseudo-HIV to test-activate tat-dependentHSVTK vectors.

1. The His^(r) expression vector pBamHis was linearized with Bam HI andtreated with calf intestinal phosphatase.

2. The Sac I site of pCV-1 was mutagenized to a Bam HI site and the 350bp Bam HI coding sequence of HIV tat was isolated.

3. The fragments purified in steps 1 and 2 were mixed, ligated, used totransform bacteria, and clones with tat in both orientations (expressingtat or the "anti-sense" tat) were identified by restriction enzymeanalysis.

These constructs were used to generate infectious recombinant vectorparticles in conjunction with a packaging cell line such as PA317, asdescribed above. These vectors are genetically stable and result inpredictable proviral structure as judged by Southern blot analysis ofrestriction-enzyme-digested genomic DNA from individual clones ofinfected cells (39/40 clones tested had proviruses of the expectedsize).

The biological properties of these retroviral vectors are describedhereinafter. The HIV tat gene ("tathis" vector--see FIG. 11) wastransfected into mouse PA317 cells. Five individual histidinol-resistantsubclones were obtained (TH 1-5) which express HIV tat. These cells arethus an experimental model for HIV infection. The vectors KTVIHAX,KTVIH5NEO, and MHMTKNEO, were subsequently introduced by infection intothese tat-expressing cell lines as well as their parent cell linelacking tat. Cell viability was then determined in variousconcentrations of the HSVTK-specific cytotoxic drug, acyclovir (ACV).The data are reported here as LD50 (the drug concentration at which 50%toxicity is observed). The parental cell line containing the vector butlacking tat (non-HIV-infected model) showed no detectable toxicity byACV at the concentrations tested (see FIG. 12). These cells thus require100 uM ACV or greater for cytotoxicity. This is true also for thesecells lacking the vectors. Thus the vectors alone, ACV alone, or eventhe vector +ACV (solid boxes) is not cytotoxic. However, cell lineswhich express HIV tat (the experimental representation of an HIVinfection) are effectively killed by ACV. This is true to varyingdegrees for all three vectors tested. These data indicate thatHIV-infected cells will be killed in the presence of ACV and"potentiator" vectors.

In an analogous experiment, vectors KTVIHAX and KTVIH5 Neo wereintroduced by infection into human T-cell and monocyte cell lines SupT1, HL60 and U937 cells. Subsequently, these cells were infected withtat his or αtat vectors, selected in histidinol, and cell viabilitydetermined at various concentrations of the ACV analog, FIAU. The LD₅₀reported in Table 1 (below) indicate that a vector dependent increase inFIAU toxicity occurs in the absence of HIV tat but is increased anadditional ten- to twentyfold when tat is present. This indicates thatalthough there is a baseline HSVTK expression in all but HL60 cells,expression is even greater in the presence of HIV tat.

                  TABLE 1    ______________________________________    HIV tat inducibility of FIAU cytotoxicity in human    monocyte and T-cell lines infected with conditionally    lethal recombinant retroviral vectors    Cell Type   Vectors    tat    LD50FIAU (μM)    ______________________________________    HL60        --         -      50    ("monocyte")                --         +      50                KTVIHAX    -      50                KTVIHAX    +      <0.2                KTVIH5Neo  -      50                KTVIH5Neo  +      <0.2    U937        --         -      10    ("monocyte")                KTVIHAX    -      0.5                KTVIHAX    +      0.05                KTVIH5Neo  -      0.5                KTVIH5Neo  +      0.05    Sup T1      --         -      10    ("T-cell")  --         +      5                KTVIHAX    -      0.5                KTVIHAX    +      0.05                KTVIH5Neo  -      0.5                KTVIH5Neo  +      0.05    H9          --         -      10    ("T-cell")  KTVIHAX    -      2                KTVIHAX    +      0.2                KTVIH5Neo  -      1                KTVIH5Neo  +      0.05    ______________________________________

Similarly, HIV infection of human T-cell line H9 +/- FIAU show afivefold preferential inhibition (through cell killing) of HIVinfection. Cultures were first treated with vector, then challenged withHIV for 4 days. Viral supernatants were then titred using the HIV assay,as described in Section IV.

In the case of HIV-infected cells, expression of the conditionallylethal HSVTK gene may be made even more HIV-specific by includingcis-acting elements in the transcript ("CRS/CAR"), which require anadditional HIV gene product, rev, for optimal activity (Rosen et al.,Proc. Natl. Acad. Sci. USA 85:2071, 1988). Such a tat- andrev-responsive vector (RRKTVIH) has been constructed (see FIG. 10) andamphotropic virus has been generated. More generally, cis elementspresent in mRNAs have been shown in some cases to regulate mRNAstability or translatability. Sequences of this type (i.e.,post-transcriptional regulation of gene expression) may be used forevent- or tissue-specific regulation of vector gene expression. Inaddition, multimerization of these sequences (i.e., rev-responsive"CRS/CAR" or tat-responsive "TAR" elements for HIV) could result in evengreater specificity. It should be noted that this kind of conditionalactivation of an inactive precursor into an active product in cells mayalso be achieved using other viral vectors with a shorter term effect,e.g., adenovirus vectors. Such vectors are capable of efficientlyentering cells and expressing proteins encoded bv the vector over aperiod of time from a couple of days to a month or so. This period oftime should be sufficient to allow killing of cells which are infectedby both HIV and the recombinant virus, leading to HIV dependentactivation of expression of a gene carried by the recombinant virus.This gene expression would then allow conversion of an inactiveprecursor into an active (e.g., lethal) product.

Production, concentration and storage of vector preparations is aspreviously described. Administration is by direct in vivo administrationas before or by ex corpore treatment of PBL and/or bone marrow. Doseswill be at approximately the same levels as for Example 4. Targeting ofviral vector infection will not be through the CD4 receptor, but may beaccomplished through producing vector particles which will infect cellsusing the HIV env protein (gp120) as a receptor. Such HIV-tropic virusesmay be produced from an MLV-based packaging cell line constructed fromcells which have naturally high levels of CD4 protein in their cellmembrane (for example, Sup T1 cells) or from any cell type "engineered"to express the protein. The resultant virions, which form by buddingfrom the cell membrane itself, contain the CD4 protein in theirmembrane. Since membranes containing CD4 are known to fuse withmembranes carrying HIV env, these virions should fuse with cellscontaining HIV env and result in the specific infection of HIV-infectedcells which have gp120 on their surface. Such a packaging cell line mayrequire the presence of an MLV env protein to allow proper virionassembly and budding to result in infectious virions. If so, an MLV envwhich does not infect human cells (such as ecotropic env) would be usedsuch that viral entry will occur only through the CD4/HIV envinteraction and not through the MLV env cell receptor, which wouldpresumably not depend upon the presence of HIV-env for infection.Alternatively, the requirement for MLV env may be satisfied by a hybridenvelope where the amino-terminal binding domain has been replaced bythe amino-terminal HIV-env binding domain of CD4. This inversion of thenormal virus-receptor interaction can be used for all types of viruseswhose corresponding cellular receptor has been identified.

In a similar manner to the preceding embodiment, the retroviral vectorconstruct can carry a gene for phosphorylation, phosphoribosylation,ribosylation, or other metabolism of a purine- or pyrimidine-based drug.This gene may have no equivalent in mammalian cells and might come fromorganisms such as a virus, bacterium, fungus, or protozoan. An exampleof this would be the E. coli guanine phosphoribosyl transferase geneproduct, which is lethal in the presence of thioxanthine (see Besnard etal., Mol. Cell. Biol. 7:4139-4141, 1987). Conditionally lethal geneproducts of this type have potential application to many presently knownpurine- or pyrimidine-based anticancer drugs, which often requireintracellular ribosylation or phosphorylation in order to becomeeffective cytotoxic agents. The conditionally lethal gene product couldalso metabolize a nontoxic drug, which is not a purine or pyrimidineanalogue, to a cytotoxic form (see Searle et al., Brit. J. Cancer53:377-384, 1986).

Mammalian viruses in general tend to have "immediate early" genes whichare necessary for subsequent transcriptional activation from other viralpromoter elements. Gene products of this nature are excellent candidatesfor intracellular signals (or "identifying agents") of viral infection.Thus, conditionally lethal genes from transcriptional promoter elementsresponsive to these viral "immediate early" gene products couldspecifically kill cells infected with any particular virus.Additionally, since the human α and β interferon promoter elements aretranscriptionally activated in response to infection by a wide varietyof nonrelated viruses, the introduction of vectors expressing aconditionally lethal gene product like HSVTK, for example, from theseviral-responsive elements (VRE) could result in the destruction of cellsinfected with a variety of different viruses.

In a fourth embodiment, the recombinant retrovirus carries a genespecifying a product which is not in itself toxic, but when processed ormodified by a protein, such as a protease specific to a viral or otherpathogen, is converted into a toxic form. For example, the recombinantretrovirus could carry a gene encoding a proprotein for ricin A chain,which becomes toxic upon processing by the HIV protease. Morespecifically, a synthetic inactive proprotein form of the toxic ricin ordiphtheria A chains could be cleaved to the active form by arranging forthe HIV virally encoded protease to recognize and cleave off anappropriate "pro" element.

In a fifth embodiment, the retroviral construct may express a "reportingproduct" on the surface of the target cells in response to the presenceof an identifying agent in the cells (such as HIV tat protein). Thissurface protein can be recognized by a cytotoxic agent, such asantibodies for the reporting protein or by cytotoxic T-cells. In asimilar manner, such a system can be used as a detection system (seebelow) to simply identify those cells having a particular gene whichexpresses an identifying protein, such as the HIV tat gene.

Similarly, in a sixth embodiment, a surface protein could be expressedwhich would itself be therapeutically beneficial. In the particular caseof HIV, expression of the human CD4 protein specifically in HIV-infectedcells may be beneficial in two ways:

1. Binding of CD4 to HIV env intracellularly could inhibit the formationof viable viral particles much as soluble CD4 has been shown to do forfree virus, but without the problem of systematic clearance and possibleimmunogenicity, since the protein will remain membrane bound and isstructurally identical to endogenous CD4 (to which the patient should beimmunologically tolerant).

2. Since the CD4/HIV env complex has been implicated as a cause of celldeath, additional expression of CD4 (in the presence of excess HIV-envpresent in HIV-infected cells) leads to more rapid cell death and thusinhibits viral dissemination. This may be particularly applicable tomonocytes and macrophages, which act as a reservoir for virus productionas a result of their relative refractility to HIV-induced cytotoxicity(which, in turn, is apparently due to the relative lack of CD4 on theircell surfaces).

EXAMPLE 6 Construction of p4TVIHAX retroviral vector (see FIG. 13)

1. The 9.4 kb Asu II/Xho I fragment was isolated from pN2.

2. The 0.6 kb Xho I/Eco R1 HIV promoter fragment was isolated frompSKHL.

3. The 1.4 kb coding region for human CD4 was isolated as an Eco R1/BstY1 (Xho II) fragment from the expression vector, pMV7T4.

4. The (A)n signal of HSVTK was isolated as a 0.3 kb Apa I/Sma Ifragment, 3' repaired with T4 polymerase and dNTP's and cloned into theSma I site of pUC31. After transforming bacteria, clones were screenedfor orientation by restriction enzyme analysis (p31 A!n +/-!). The 0.3kb (A)n signal was then isolated as a 0.3 kb Bgl II/Acc I fragment.

5. 1-4 clones were mixed, ligated, used to transform bacteria and cloneswere identified by restriction enzyme analysis.

Recombinant amphotropic retroviruses have been produced and introducedinto human monocyte and T-cell lines lacking or containing the HIV tatexpression vector, tathis. Syncytia assays with HIV env-expressing mousefibroblasts show that monocyte cell lines HL60 and U937 themselves lacksufficient CD4 to fuse with these cells. However, HL60 and U937 cellscontaining vector 4TVIHAX can fuse with the reporter cells (HIV-envexpressing cells) when HIV tat is present, but not in its absence. Thesedata indicate that CD4 expression is inducible and biologically active(as judged by syncytia formation). Experiments with the vector in humanT-cell line, H9, indicated exceptionally high toxicity due to HIVinfection and a correspondingly low HIV titre (more than 200-fold lowerthan the HIV titre produced in H9 cells lacking the vector).

In a seventh embodiment, the retroviral vector codes for a ribozymewhich will cleave and inactivate RNA molecules essential for viabilityof the vector infected cell. By making ribozyme production dependent onan intracellular signal corresponding to the pathogenic state, such asHIV tat, toxicity is specific to the pathogenic state.

IV. Immune Down-Regulation

As briefly described above, the present invention provides recombinantretroviruses which carry a vector construct capable of suppressing oneor more elements of the immune system in target cells infected with theretrovirus.

Specific down-regulation of inappropriate or unwanted immune responses,such as in chronic hepatitis or in transplants of heterologous tissuesuch as bone marrow, can be engineered using immune-suppressive viralgene products which suppress surface expression of transplantation (MHC)antigen. Group C adenoviruses Ad2 and Ad5 possess a 19 kd glycoprotein(gp 19) encoded in the E3 region of the virus. This gp 19 molecule bindsto class I MHC molecules in the endoplasmic reticulum of cells andprevents terminal glycosylation and translocation of class I MHC to thecell surface. For example, prior to bone marrow transplantation, donorbone marrow cells may be infected with gp 19-encoding vector constructswhich upon expression of the gp 19 inhibit the surface expression of MHCclass I transplantation antigens. These donor cells may be transplantedwith low risk of graft rejection and may require a minimalimmunosuppressive regimen for the transplant patient. This may allow anacceptable donor-recipient chimeric state to exist with fewercomplications. Similar treatments may be used to treat the range ofso-called autoimmune diseases, including lupus erythromatosis, multiplesclerosis, rheumatoid arthritis or chronic hepatitis B infection.

An alternative method involves the use of anti-sense message, ribozyme,or other specific gene expression inhibitor specific for T-cell cloneswhich are autoreactive in nature. These block the expression of theT-cell receptor of particular unwanted clones responsible for anautoimmune response. The anti-sense, ribozyme, or other gene may beintroduced using the viral vector delivery system.

V. Expression of Markers

The above-described technique of expressing a palliative in a cell, inresponse to some identifying agent, can also be modified to enabledetection of a particular gene in a cell which expresses an identifyingprotein (for example, a gene carried by a particular virus), and henceenable detection of cells carrying that virus. In addition, thistechnique enables the detection of viruses (such as HIV) in a clinicalsample of cells carrying an identifying protein associated with thevirus.

This modification can be accomplished by providing a genome coding for aproduct, the presence of which can be readily identified (the "markerproduct"), and carrying a promoter, which responds to the presence ofthe identifying protein in indicator cells, by switching expression ofthe reporting product between expressing and nonexpressing states. Forexample, HIV, when it infects suitable indicator cells, makes tat andrev. The indicator cells can thus be provided with a genome (such as byinfection with an appropriate recombinant retrovirus) which codes for amarker gene, such as the alkaline phosphatase gene, β-galactosidase geneor the luciferase gene, and a promoter, such as the HIV promoter, whichcontrols expression of the marker gene. When the indicator cells areexposed to a clinical sample to be tested, and the sample contains HIV,the indicator cells become infected with HIV, resulting in tat and/orrev expression (an identifying protein) therein. The HIV expressioncontrols in the indicator cells would then respond to tat and/or revproteins by switching expression of genes encoding β-galactosidase,luciferase, or alkaline phosphatase (marker products) from normally"off" to "on." In the case of β-galactosidase or alkaline phosphatase,exposing the cells to substrate analogues results in a color orfluorescence change if the sample is positive for HIV. In the case ofluciferase, exposing the sample to luciferin will result in luminescenceif the sample is positive for HIV. For intracellular enzymes such asβ-galactosidase, the viral titre can be measured directly by countingcolored or fluorescent cells, or by making cell extracts and performinga suitable assay. For the membrane bond form of alkaline phosphatase,virus titre can also be measured by performing enzyme assays on the cellsurface using a fluorescent substrate. For secreted enzymes, such as anengineered form of alkaline phosphatase, small samples of culturesupernatant are assayed for activity, allowing continuous monitoring ofa single culture over time. Thus, different forms of this marker systemcan be used for different purposes. These include counting active virusor sensitively and simply measuring viral spread in a culture and theinhibition of this spread by various drugs.

Further specificity can be incorporated into the preceding system bytesting for the presence of the virus either with or withoutneutralizing antibodies to that virus. For example, in one portion ofthe clinical sample being tested, neutralizing antibodies to HIV may bepresent; whereas in another portion there would be no neutralizingantibodies. If the tests were negative in the system where there wereantibodies and positive where there were no antibodies, this wouldassist in confirming the presence of HIV.

Within an analogous system for an in vitro assay, the presence of aparticular gene, such as a viral gene, may be determined in a cellsample. In this case, the cells of the sample are infected with asuitable retroviral vector which carries the reporter gene linked to theexpression controls of the virus of interest. The reporter gene, afterentering the sample cells, will express its reporting product (such asβ-galactosidase or luciferase) only if the host cell expresses theappropriate viral proteins.

These assays are more rapid and sensitive, since the reporter gene canexpress a greater amount of reporting product than identifying agentpresent, which results in an amplification effect. Example 7 describes arepresentative technique for detecting a gene which expresses anidentifying protein.

EXAMPLE 7 HIV-Specific Marker System or Assay

A. Constructs

Reporter constructs under the control of the HIV expression system areshown in FIG. 14 (a recombinant retroviral vector) and in FIG. 15 (asimple plasmid used by transfection). The pieces of these preferredvector and plasmid reporters were derived as follows:

The retroviral backbone was derived from the construct pAFVXM (Kriegeret al., Cell 38:384, 1984), which had been linearized using Xho I andCla I. SV₂ neo was obtained from the plasmid pKoneo (Hanahan, unpubl.)by isolation of the 1.8 kb Cla I fragment.

The HIV LTR was isolated as a 0.7 kb Hind III fragment from the plasmidpC15CAT (Arya et al., Science 229:69, 1985). Beta-gal was obtained fromthe plasmid pSP65 β-gal (Cepko, pers. comm.) as a Hind III-Sma Ifragment. A secreted form of human placental alkaline phosphatase wasproduced by introduction of a universal terminator sequence afteramino-acid 489 of the cell surface form of alkaline phosphatase (asdescribed by Berger et al., Gene 66:1, 1988). The secreted alkalinephosphatase gene was isolated as a 1.8 kb Hind III to Kpn I fragment.The CRS-CAR sequences from HIV env were obtained by isolating the 2.1 kbKpn I to Bam HI fragment from HTLVIIIB/BH1OR3 (Fisher et al., Science233:655, 1986). This fragment was inserted into pUC31 linearized by BamHI, and Kpn I pUC31 is pUC19 (Yanisch-Perron et al., Gene 33:103, 1985)with extra Xho I, Bgl II, Bssh II and Nco I sites between the Eco R1 andKpn I sites of pUC19. The Bam HI site of the resulting construct wasconverted to a Nco I site to allow resection of the CRS-CAR sequences byNco I digestion. The SV40 t intron was obtained from pSVOL (de Wet etal., Mol. Cell. Biol. 7:725, 1987) as a 0.8 kb Nco I to Bam HI fragment.

B. Indicator Cells and Retroviral Vectors

Human T-cell (H-9, CEM and Sup T1) and monocyte (U-937) cell lines wereobtained from ATCC, and maintained in RPM1 1640 medium supplemented with10% fetal bovine serum and 1% penicillin/streptomycin.

The nonretroviral vectors were introduced into cell lines byelectroporation using a Bio-Rad Gene Pulser. The cell lines wereselected in G-418 (1 mg/ml) for 2-3 weeks to obtain stable G-418_(R)cell lines, and then dilution cloned to obtain clonal cell lines.

The pAF vectors were transfected into the PA317 packaging cell line as acalcium phosphate precipitate (Wigler et al., Cell 16:777, 1979). Thevirus-producing PA317 cells were co-cultivated with human monocyte celllines for 24 hours in the presence of polybrene, after which thesuspension cells were removed and selected in G-418 and subcloned asabove.

C. Assay

Stable cell lines were infected with HIV (HTLV III_(B)) and the cells(β-gal) or media (alkaline phosphatase) assayed on a daily basis for 6days post-infection.

β-Galactosidase Assay

Infected cells could be assayed by either: (i) In situ histochemicalstaining as described by MacGregor et al. Somatic Cell and Mol. Genetics13:253, 1987); or (ii) by using cell extracts in a solution enzymaticassay with ONPG as a substrate (Norton and Coffin, Mol. Cell. Biol.5:281, 1985).

Soluble Alkaline Phosphatase Assay

Medium was removed from infected cells, microfuged for 10 seconds, andthen heated to 68° C. for 10 minutes to destroy endogenous phosphatases.The medium was then microfuged for 2 minutes and an aliquot (10-50 μl)removed for assay. 100 μl of buffer (1M diethanolamine, pH 9.8; 0.5 MmMgCl₂ ; 10 mM L-homoarginine) was added and then 20 μl of 120 mMp-nitrophenylphosphate (in buffers) was added. The A₄₀₅ of the reactionmixture was monitored using an automatic plate reader.

FIGS. 16 and 17 depict typical results of a time course of infection ofSup T1 cells using the alkaline phosphatase assay in the presence ofvarying concentrations of antiviral drugs. The "+" and "-" on day 6indicate the presence or absence of syncytia.

The present invention provides a number of other techniques (describedbelow) which can be used with the retroviral vector systems employedabove, so as to enhance their performance. Alternatively, thesetechniques may be used with other gene-delivery systems.

VI. Packaging Cell Selection

This aspect of the present invention is based, in part, upon thediscovery of the major causes of low recombinant virus titres frompackaging cells, and of techniques to correct those causes. Basically,at least five factors may be postulated as causes for low recombinantvirus titres:

1. the limited availability of viral packaging proteins;

2. the limited availability of retroviral vector RNA genomes;

3. the limited availability of cell membrane for budding of therecombinant retroviruses;

4. the limited intrinsic packaging efficiency of the retroviral vectorgenome; and

5. the density of the receptor specific for the envelope of a givenretrovirus.

As noted above, the limited availability of viral packaging proteins isthe initial limiting factor in recombinant retrovirus production frompackaging cells. When the level of packaging protein in the packagingcells is increased, titre increases to about 10⁵ infectiousunits/milliliter, following which increasing packaging protein level hasno further effect on titres. However, titres can be further augmented byalso increasing the level of retroviral vector genome available forpackaging. Thus, as described herein, it is advantageous to selectproducer cells that manufacture the maximum levels of packaging proteinsand retroviral vector genomes. It has been discovered that the methodsof identifying, and thus selecting, packaging cells and producer cells,described earlier under the section entitled "Background of theInvention," tend to lead to selection of many producer cells whichproduce low titres for the reasons described below.

The present invention takes advantage of the previously disadvantageousfact that the protein expression level of a gene downstream from the 5'LTR or other promoter, and spaced therefrom by an intervening gene, issubstantially less than if the intervening gene were absent. In thepresent invention, the selectable gene is placed downstream from a geneof the packaging genome or the gene of interest carried by the vectorconstruct, but is still transcribed under the control of the viral 5'LTR or other promoter without any splice donor or splice acceptor sites.This accomplishes two things. First, since the packaging genes or genesof interest are now upstream with no intervening gene between themselvesand the promoter, their corresponding proteins (packaging protein orprotein of interest) will be expressed at a higher level (five- totwentyfold) than the selectable protein. Second, the selectable proteinwill be expressed on average at a lower level, with the distribution oflevel of expression shifting toward lower levels. In the case of thephleor protein, this shift in distribution is illustrated by the brokencurve indicated in FIG. 18. However, the selection level for resistanceto phleomycin remains the same, so that only the top-end expressingcells survive. The levels of the packaging protein or of the protein ofinterest will still be proportional, only in this case, a higher levelof selectable protein corresponds to a much higher level of packagingprotein or protein of interest.

Preferably, the foregoing procedure is performed using a plasmidcarrying one of the proviral gag/pol or env packaging genes, along witha first selectable gene. These cells are then screened for the cellsproducing the highest levels of protein by reaction with an antibodyagainst env (or possibly gag/pol), a second fluorescent antibody, andthen sorted on a fluorescence-activated cell sorter (FACS).Alternatively, other tests for protein level may be used. Subsequently,the procedure and screening are repeated using those selected cells, andthe other of the gag/pol or env packaging genes. In this step, a secondselectable gene (different from the first) would be required downstreamfrom the packaging gene and the cells producing the largest amount ofthe second viral protein selected. The procedure and screening are thenrepeated using the surviving cells, with a plasmid carrying the proviralvector construct bearing the gene of interest and a third selectablegene, different from the first or second selectable gene. As a result ofthis procedure, cells producing high titres of the desired recombinantretrovirus will be selected, and these can be cultured as required tosupply recombinant retrovirus. In addition, gag and pol can beindependently introduced and selected.

Example 8 describes the construction of gag/pol and env plasmidsdesigned to use these procedures.

EXAMPLE 8 Plasmids Designed to Make High Levels of Packaging Proteins(FIG. 19)

1. The 2.7 kb Xba I fragment from pPAM (Miller et al., Mol. Cell. Biol.5:431, 1985), which contains the amphotrophic env segment, was cloned inpUC18 at the Xba I site, then removed with Hind III and Sma I. Thisfragment was cloned into the vector pRSV neo (Gorman et al., Mol. Cell.Biol. 2:1044, 1982; Southern et al., J. Mol. Appl. Genet. 1:327, 1982)cut with Hind III and Pvu II, to give pRSV env. A 0.7 kb Bam HI to BstEII fragment from the plasmid pUT507 (Mulsant et al., Somat. Cell. Mol.Genet. 14:243, 1988) with the BstE II end filled in carries the phleoresistance coding sequence. The 4.2 kb Bam HI to Xho I fragment, thecontiguous 1.6 kb Xho I to Xba I (Xba I filled in) from RSVenv, and thephleo fragment were ligated to give pRSVenv-phleo.

2. A fragment from the Pst I site at nucleotide 563 of MLV (RNA TumorViruses, Vol. II, Cold Spring Harbor, 1985) to the Sca I site at 5870was derived from pMLV-K (Miller et al., 1985, op. cit.) and cloned inthe Pst I to Bam HI (Bam HI filled-in) fragment from p4aA8 (Jolly etal., Proc. Natl. Acad. Sci. USA 80:477, 1983) that has the SV40promoter, the pBR322 ampicillin resistance and origin of replication andthe SV40 poly A site. This gives pSVgp. pSVgpDHFR was made using thefollowing fragments: the 3.6 kb Hind III to Sal I fragment from pSVgpcontaining the SV40 promoter plus MLV gag and some pol sequences; the2.1 kb Sal I to Sca I fragment from pMLV-K with the rest of the polgene, the 3.2 kb Xba I (Xba I filled-in) to Pst I fragment from pF400with the DHFR gene plus poly A site, pBR322 origin and half theampicillin resistance gene; the 0.7 kb Pst I to Hind III fragment frompBR322 with the other half of the ampicillin resistance gene. This givespSVgp-DHFR. All these constructs are shown in FIG. 19. These plasmidscan be transfected into 3T3 cells or other cells and high levels of gag,pol or env obtained.

An additional method for accomplishing selection is to use a geneselection in one round and its antisense in a subsequent round. Forexample, gag/pol may be introduced into an HPRT-deficient cell with theHPRT gene and selected for the presence of this gene using that mediawhich requires HPRT for the salvage of purines. In the next round, theantisense to HPRT could be delivered downstream to env and the cellselected in 6 thioguanine for the HPRT-deficient phenotype. Largeamounts of antisense HPRT would be required in order to inactivate theHPRT gene transcripts, assuming no reversion occurred.

In addition to the gag/pol expressing constructs which begin atnucleotide 563 of MoMLV, several others can be constructed which containupstream lead sequences. It has been observed by Prats et al. (RNA TumorViruses Meetinq, Cold Spring Harbor, N.Y., 1988) that a glycosylatedform of the gag protein initiates at nucleotide 357 and a translationenhancer maps in the region between nucleotides 200-270. Therefore,gag/pol expressing constructs may be made beginning at the Bal I site(nucleotide 212) or Eag I site (nucleotide 346) to include theseupstream elements and enhance vector production.

Envelope Substitutions

The ability to express gag/pol and env function separately allows formanipulation of these functions independently. A cell line thatexpresses ample amounts of gag/pol can be used, for example, to addressquestions of titre with regard to env. One factor resulting in lowtitres is the density of appropriate receptor molecules on the targetcell or tissue. A second factor is the affinity of the receptor for theviral envelope protein. Given that env expression is from a separateunit, a variety of envelope genes (requiring different receptorproteins), such as xenotropic, polytropic, or amphotrophic envs from avariety of sources, can be tested for highest titres on a specifictarget tissue. Furthermore, envelopes from nonmurine retrovirus sourcescan be used for pseudotyping a vector. The exact rules for pseudotyping(i.e., which envelope proteins will interact with the nascent vectorparticle at the cytoplasmic side of the cell membrane to give a viableviral particle (Tato, Virology 88:71, 1978) and which will not (Vana,Nature 336:36, 1988), are not well characterized. However, since a pieceof cell membrane buds off to form the viral envelope, molecules normallyin the membrane are carried along on the viral envelope. Thus, a numberof different potential ligands can be put on the surface of viralvectors by manipulating the cell line making gag and pol in which thevectors are produced or choosing various types of cell lines withparticular surface markers. One type of surface marker that can beexpressed in helper cells and that can give a useful vector-cellinteraction is the receptor for another potentially pathogenic virus.The pathogenic virus displays on the infected cell surface its virallyspecific protein (e.g., env) that normally interacts with the cellsurface marker or receptor to give viral infection. This reverses thespecificity of the infection of the vector with respect to thepotentially pathogenic virus by using the same viral protein-receptorinteraction, but with the receptors on the vector and the viral proteinon the cell.

It may be desirable to include a gene which encodes for an irrelevantenvelope protein which does not lead to infection of target cells by thevector so produced, but does facilitate the formation of infectiousviral particles. For example, one could use human Sup Tl cells as ahelper line. This human T-cell line expresses CD4 molecules at highlevels on its surface. Conversion of this into a helper line can beachieved by expressing gag/pol with appropriate expression vectors andalso, if necessary, the Moloney ecotropic env gene product as anirrelevant (for human cells) envelope protein (the Moloney ecotropic envonly leads to infection of mouse cells) . Vectors produced from such ahelper line would have CD4 molecules on their surfaces and are capableof infecting only cells which express HIV env, such as HIV-infectedcells.

In addition, hybrid envelopes (as described below) can be used in thissystem as well, to tailor the tropism (and effectively increase titres)of a retroviral vector. A cell line that expresses ample amounts of agiven envelope gene can be employed to address questions of titre withregard to gag and pol.

Cell Lines

The most common packaging cell lines used for MOMLV vector systems(psi2, PA12, PA317) are derived from murine cell lines. There areseveral reasons why a murine cell line is not the most suitable forproduction of human therapeutic vectors:

1. They are known to contain endogenous retroviruses.

2. They contain nonretroviral or defective retroviral sequences that areknown to package efficiently.

3. There may be deleterious effects caused by the presence of murinecell membrane components.

Several non-murine cell lines are potential packaging lines. Theseinclude Vero cells which are used in Europe to prepare polio vaccine,WI38 which are used in the U.S. in vaccine production, CHO cells whichare used in the U.S. for TPA preparation and D17 or other dog cells thatmay have no endogenous viruses.

Although the factors that lead to efficient infection of specific celltypes by retroviral vectors are not completely understood, it is clearthat because of their relatively high mutation rate, retroviruses may beadapted for markedly improved growth in cell types in which initialgrowth is poor, simply by continual reinfection and growth of the virusin that cell type (the adapter cell). This can also be achieved usingviral vectors that encode some viral functions (e.g., env), and whichare passed continuously in cells of a particular type which have beenengineered to have the functions necessary to complement those of thevector to give out infectious vector particles (e.g., gag/pol). Forexample, one can adapt the murine amphotropic virus 4070A to humanT-cells or monocytes by continuous growth and reinfection of eitherprimary cell cultures or permanent cell lines such as Sup T1 (T-cells)or U937 (monocytes). Once maximal growth has been achieved, as measuredby reverse transcriptase levels or other assays of virus production, thevirus is cloned out by any of a number of standard methods, the clone ischecked for activity (i.e., the ability to give the same maximal growthcharacteristic on transfection into the adapter cell type) and thisgenome used to make defective helper genomes and/or vectors which inturn, in an appropriately manufactured helper or producer line, willlead to production of viral vector particles which infect and express inthe adapter cell type with high efficiency (10⁸ -10⁹ infectiousunits/ml).

VII. Alternative Viral Vector Packaging Techniques

Two additional alternative systems can be used to produce recombinantretroviruses carrying the vector construct. Each of these systems takesadvantage of the fact that the insect virus, baculovirus, and themammalian viruses, vaccinia and adenovirus, have been adapted recentlyto make large amounts of any given protein for which the gene has beencloned. For example, see Smith et al. (Mol. Cell. Biol. 3:12, 1983);Piccini et al. (Meth. Enzvmoloay, 153:545, 1987); and Mansour et al.(Proc. Natl. Acad. Sci. USA 82:1359, 1985).

These viral vectors can be used to produce proteins in tissue culturecells by insertion of appropriate genes into the viral vector and,hence, could be adapted to make retroviral vector particles.

Adenovirus vectors are derived from nuclear replicating viruses and canbe defective. Genes can be inserted into vectors and used to expressproteins in mammalian cells either by in vitro construction (Ballay etal., EMBO J. 4:3861, 1985) or by recombination in cells (Thummel et al.,J. Mol. Appl. Genetics 1:435, 1982).

One preferred method is to construct plasmids using the adenovirus MajorLate Promoter (MLP) driving: (1) gag/pol, (2) env, (3) a modified viralvector construct. A modified viral vector construct is possible becausethe U3 region of the 5' LTR, which contains the viral vector promoter,can be replaced by other promoter sequences (see, for example, Hartman,Nucl. Acids Res. 16:9345, 1988). This portion will be replaced after oneround of reverse transcriptase by the U3 from the 3' LTR.

These plasmids can then be used to make adenovirus genomes in vitro(Ballay et al., op. cit.), and these transfected in 293 cells (a humancell line making adenovirus ElA protein), for which the adenoviralvectors are defective, to yield pure stocks of gag/pol, env andretroviral vector carried separately in defective adenovirus vectors.Since the titres of such vectors are typically 10⁷ -10¹¹ /ml, thesestocks can be used to infect tissue culture cells simultaneously at highmultiplicity. The cells will then be programmed to produce retrdviralproteins and retroviral vector genomes at high levels. Since theadenovirus vectors are defective, no large amounts of direct cell lysiswill occur and retroviral vectors can be harvested from the cellsupernatants.

Other viral vectors such as those derived from unrelated retroviralvectors (e.g., RSV, MMTV or HIV) can be used in the same manner togenerate vectors from primary cells. In one embodiment, these adenoviralvectors are used in conjunction with primary cells, giving rise toretroviral vector preparations from primary cells.

In an alternative system (which is more truly extracellular), thefollowing components are used:

1. gag/pol and env proteins made in the baculovirus system in a similarmanner as described in Smith et al. (supra) (or in other proteinproduction systems, such as yeast or E. coli);

2. viral vector RNA made in the known T7 or SP6 or other in vitroRNA-generating system (see, for example, Flamant and Sorge, J. Virol.62:1827, 1988);

3. tRNA made as in (2) or purified from yeast or mammalian tissueculture cells;

4. liposomes (with embedded env protein); and

5. cell extract or purified necessary components (when identified)(typically from mouse cells) to provide env processing, and any or othernecessary cell-derived functions.

Within this procedure (1), (2) and (3) are mixed, and then env protein,cell extract and preliposome mix (lipid in a suitable solvent) added. Itmay, however, be necessary to earlier embed the env protein in theliposomes prior to adding the resulting liposome-embedded env to themixture of (1), (2), and (3). The mix is treated (e.g., by sonication,temperature manipulation, or rotary dialysis) to allow encapsidation ofthe nascent viral particles with lipid plus embedded env protein in amanner similar to that for liposome encapsidation of pharmaceuticals, asdescribed in Gould-Fogerite et al., Anal. Biochem. 148:15, 1985). Thisprocedure allows the production of high titres of replicationincompetent recombinant retroviruses without contamination withpathogenic retroviruses or replication-competent retroviruses.

VIII. Cell Line-Specific Retroviruses--"Hybrid Envelope"

The host cell range specificity of a retrovirus is determined in part bythe env gene products. For example, Coffin, J. (RNA Tumor Viruses2:25-27, Cold Spring Harbor, 1985) notes that the extracellularcomponent of the proteins from murine leukemia virus (MLV) and RousSarcoma virus (RSV) are responsible for specific receptor binding. Thecytoplasmic domain of envelope proteins, on the other hand, areunderstood to play a role in virion formation. While pseudotyping (i.e.,the encapsidation of viral RNA from one species by viral proteins ofanother species) does occur at a low frequency, the envelope protein hassome specificity for virion formation of a given retrovirus. The presentinvention recognizes that by creating a hybrid env gene product (i.e.,specifically, an env protein having cytoplasmic regions and exogenousbinding regions which are not in the same protein molecule in nature)the host range specificity may be changed independently from thecytoplasmic function. Thus, recombinant retroviruses can be producedwhich will specifically bind to preselected target cells.

In order to make a hybrid protein in which the receptor bindingcomponent and the cytoplasmic component are from different retroviruses,a preferred location for recombination is within the membrane-spanningregion of the cytoplasmic component. Example 9 describes theconstruction of a hybrid env gene which expresses a protein with the CD4binding portion of the HIV envelope protein coupled to the cytoplasmicdomain of the MLV envelope protein.

EXAMPLE 9 Hybrid HIV-MLV Envelopes

A hybrid envelope gene is prepared using in vitro mutagenesis (Kunkel,Proc. Natl. Acad. Sci. USA 82:488-492, 1985) to introduce a newrestriction site at an appropriate point of junction. Alternatively, ifthe two envelope sequences are on the same plasmid, they can be joineddirectly at any desired point using in vitro mutagenesis. The end resultin either case is a hybrid gene containing the 5' end of the HIV gp 160and the 3' end of MLV p15E. The hybrid protein expressed by theresulting recombinant gene is illustrated in FIG. 20 and contains theHIV gp120 (CD4 receptor binding protein), the extracellular portion ofHIV gp 41 (the gp 120 binding and fusigenic regions), and thecytoplasmic portion of MLV p15E, with the joint occurring at any ofseveral points within the host membrane. A hybrid with a fusion joint atthe cytoplasmic surface (joint C in FIG. 20) causes syncytia whenexpressed in Sup T1 cells. The number of apparent syncytia areapproximately one-fifth that of the nonhybrid HIV envelope gene in thesame expression vector. Syncytia with the hybrid occurs only when therev protein is co-expressed in trans. A hybrid with a fusion joint atthe extracellular surface (joint A in FIG. 20) gives no syncytia whilehybrid B (in the middle of the transmembrane regions) givesapproximately five-fold less syncytium on Sup T1 cells than hybrid C.

While Example 9 illustrates one hybrid protein produced from twodifferent retroviruses, the possibilities are not limited toretroviruses or other viruses. For example, the beta-receptor portion ofhuman interleukin-2 may be combined with the envelope protein of MLV. Inthis case, a recombination would preferably be located in the gp 70portion of the MLV env gene, leaving an intact p15E protein.Furthermore, the foregoing technique may be used to create a recombinantretrovirus with an envelope protein which recognizes antibody Fcsegments. Monoclonal antibodies which recognize only preselected targetcells only could then be bound to such a recombinant retrovirusexhibiting such envelope proteins so that the retrovirus would bind toand infect only those preselected target cells.

IX. Site-Specific Integration

Targeting a retroviral vector to a predetermined locus on a chromosomeincreases the benefits of gene-delivery systems. A measure of safety isgained by direct integration to a "safe" spot on a chromosome, i.e., onethat is proven to have no deleterious effects from the insertion of avector. Another potential benefit is the ability to direct a gene to an"open" region of a chromosome, where its expression would be maximized.Two techniques for integrating retroviruses at specific sites aredescribed below.

(i) Homologous Recombination

One technique for integrating an exogenous gene of a vector construct ofa recombinant retrovirus into a specific site in a target cell's DNAemploys homologous recombination. Plasmids containing sequences of DNAof greater than about 300 bp that are homologous to genomic sequenceshave been shown to interact (either by replacement or insertion) withthose genomic sequences at a rate that is greater than 10³ -fold over aspecific interaction in the absence of such homology (see Thomas andCapecchi, Cell 51:503-12, 1987; and Doetscheman et al., Nature330:576-78, 1987). It has been shown that an insertion event, oralternatively, a replacement event, may be driven by the specific designof the vector.

In order to employ homologous recombination in site-specific retroviralintegration, a vector construct should be modified such that (a)homologous sequences (to the target cell's genome) are incorporated intothe construct at an appropriate location; and (b) the normal mechanismof integration does not interfere with the targeting occurring due tohomologous sequences. A preferred approach in this regard is to addhomologous sequences (greater than about 300 bp) in the 3' LTR,downstream from the U3 inverted repeat. In this approach, the constructis initially made with a region of homology inserted in the 3' LTR atthe Nhe 1 site in U3. Reverse transcription in the host cell will resultin a duplication of the region of homology in the 5' LTR within 31 bp ofthe end of the inverted repeat (IR). Integration into the host genomewill occur in the presence or absence of the normal integrationmechanism. The gene in the vector may be expressed, whether from the LTRor from an internal promoter. This approach has the effect of placing aregion of homology near a potential free end of the double-strandedretrovirus vector genome. Free ends are known to increase the frequencyof homologous recombination by a factor of approximately ten. In thisapproach, it may be necessary to defeat the normal mechanism ofintegration, or to at least modify it to slow down the process, allowingtime for homologous DNAs to line up. Whether this latter modification isrequired in a particular case can be readily ascertained by one skilledin the art.

(ii) Integrase Modification

Another technique for integrating a vector construct into specific,preselected sites of a target cell's genome involves integrasemodification.

The retrovirus pol gene product is generally processed into four parts:(i) a protease which processes the viral gag and pol products; (ii) thereverse transcriptase; and (iii) RNase H, which degrades RNA of anRNA/DNA duplex; and (iv) the endonuclease or "integrase."

The general integrase structure has been analyzed by Johnson et al.(Proc. Natl. Acad. Sci. USA 83:7648-7652, 1986). It has been proposedthat this protein has a zinc binding finger with which it interacts withthe host DNA before integrating the retroviral sequences.

In other proteins, such "fingers" allow the protein to bind to DNA atparticular sequences. One illustrative example is the steroid receptors.In this case, one can make the estrogen receptor, responding toestrogens, have the effect of a glucocorticoid receptor, responding toglucocorticoids, simply by substituting the glucocorticoid receptor"finger" (i.e., DNA binding segment) in place of the estrogen receptorfinger segment in the estrogen receptor gene. In this example, theposition in the genome to which the proteins are targeted has beenchanged. Such directing sequences can also be substituted into theintegrase gene in place of the present zinc finger. For instance, thesegment coding for the DNA binding region of the human estrogen receptorgene may be substituted in place of the DNA binding region of theintegrase in a packaging genome. Initially, specific integration wouldbe tested by means of an in vitro integration system (Brown et al., Cell29:347-356, 1987). To confirm that the specificity would be seen invivo, this packaging genome is used to make infectious vector particles,and infection of and integration into estrogen-sensitive andestrogen-nonsensitive cells compared in culture.

Through use of this technique, incoming viral vectors may be directed tointegrate into preselected sites on the target cell's genome, dictatedby the genome-binding properties of site-specific DNA-binding proteinsegments spliced into the integrase genome. It will be understood bythose skilled in the art that the integration site must, in fact, bereceptive to the fingers of the modified integrase. For example, mostcells are sensitive to glucocorticoids and hence their chromatin hassites for glucocorticoid receptors. Thus, for most cells, a modifiedintegrase having a glucocorticoid receptor finger would be suitable tointegrate the proviral vector construct at those glucocorticoidreceptor-binding sites.

X. Production of Recombinant Retroviral Vectors in Transgenic Animals

Two problems previously described with helper line generation ofretroviral vectors are: (a) difficulty in generating large quantities ofvectors; and (b) the current need to use permanent instead of primarycells to make vectors. These problems can be overcome with producer orpackaging lines that are generated in transgenic animals. These animalswould carry the packaging genomes and retroviral vector genomes. Currenttechnology does not allow the generation of packaging cell lines anddesired vector-producing lines in primary cells due to their limitedlife span. The current technology is such that extensivecharacterization is necessary, which eliminates the use of primary cellsbecause of senescence. However, individual lines of transgenic animalscan be generated by the methods provided herein which produce thepackaging functions, such as gag, pol or env. These lines of animals arethen characterized for expression in either the whole animal or targetedtissue through the selective use of housekeeping or tissue-specificpromoters to transcribe the packaging functions. The vector to bedelivered is also inserted into a line of transgenic animals with atissue-specific or housekeeping promoter. As discussed above, the vectorcan be driven off such a promoter substituting for the U3 region of the5' LTR (FIG. 21). This transgene could be inducible or ubiquitous in itsexpression. This vector, however, is not packaged. These lines ofanimals are then mated to the gag/pol/env animal and subsequent progenyproduce packaged vector. The progeny, which are essentially identical,are characterized and offer an unlimited source of primary producingcells. Alternatively, primary cells making gag/pol and env and derivedfrom transgenic animals can be infected or transfected in bulk withretrovirus vectors to make a primary cell producer line. Many differenttransgenic animals or insects could produce these vectors, such as mice,rats, chickens, swine, rabbits, cows, sheep, fish and flies. The vectorand packaging genomes would be tailored for species infectionspecificity and tissue-specific expression through the use oftissue-specific promoters and different envelope proteins. An example ofsuch a procedure is illustrated in FIG. 22.

Although the following examples of transgenic production of primarypackaging lines are described only for mice, these procedures can beextended to other species by those skilled in the art. These transgenicanimals may be produced by microinjection or gene transfer techniques.Given the homology to MLV sequences in mice genome, the final preferredanimals would not be mice.

EXAMPLE 10 Production of Gag/Pol Proteins Using Housekeeping Promotersfor Ubicuitous Expression in Transgenic Animals

An example of a well-characterized housekeeping promoter is the HPRTpromoter. HPRT is a purine salvage enzyme which expresses in alltissues. (See Patel et al., Mol. Cell Biol. 6:393-403, 1986 and Meltonet al., Proc. Natl. Acad. Sci. 81:2147-2151, 1984). This promoter isinserted in front of various gag/pol fragments (e.g., Bal I/Sca I; AatII/Sca I; Pst I/Sca I of MOMLV; see RNA Tumor Viruses 2, Cold SpringHarbor Laboratory, 1985) that are cloned in Bluescript plasmids(Strategene, Inc.) using recombinant DNA techniques (see Maniatis etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982).The resulting plasmids are purified (Maniatis et al., op. cit.) and therelevant genetic information isolated using Geneclean (Bio 101) orelectroelution (see Hogan et al. (eds.), Manipulating the Mouse Embryo:A Laboratory Manual, Cold Spring Harbor, 1986).

These fully characterized DNAs are microinjected in the pronucleus offertilized mouse ova at a concentration of 2 ug/ml. Live-born mice arescreened by tail blot analyses (see Hogan et al., op. cit.).Transgenic-positive animals are characterized for expression levels ofgag-pol proteins by immunoprecipitation of radiolabeled primary cells,such as fibroblast (see Harlow et al. (eds.), Antibodies: A LaboratoryManual, Cold Spring Harbor, 1988). Animals then bred to homozygosity forestablishment of animal lines that produce characterized levels ofgag-pol.

EXAMPLE 11 Production of env Proteins/Hybrid Envelope Proteins UsingHousekeeping Promoters for Ubiquitous Expression in Transgenic Animals

This example utilizes the HPRT promoter for expression of eitherenvelope or hybrid envelope proteins. The envelope proteins can be fromany retrovirus that is capable of complementing the relevant gag-pol, inthis case that of MLV. Examples are ecotropic MLV, amphotrophic MLV,xenotropic MLV, polytropic MLV, or hybrid envelopes. As above, theenvelope gene is cloned behind the HPRT promoter using recombinant DNAtechniques (see Maniatis et al., op. cit.). The resulting "minigene" isisolated (see Hogan et al., op. cit.), and expression of envelopeprotein is determined (Harlow et al., op. cit.). The transgenic envelopeanimals are bred to homozygosity to establish a well-characterizedenvelope animal.

EXAMPLE 12 Production of gag-pol-env Animals Using HousekeepingPromoters for Ubiquitous Expression in Transgenic Animals

This uses the well-characterized gag-pol animals, as well as the animalsfor the establishment of a permanent gag-pol/envelope animal line. Thisinvolves breeding to homozygosity and the establishment of awell-characterized line. These lines are then used to establish primarymouse embryo lines that can be used for packaging vectors in tissueculture. Furthermore, animals containing the retroviral vector are bredinto this line.

EXAMPLE 13 Production of Tissue-Specific Expression of gag-pol-env orHybrid Envelope in Transgenic Animals

This example illustrates high level expression of the gag/pol, envelope,or hybrid envelope in specific tissues, such as T-cells. This involvesthe use of CD2 sequences (see Lang et al., EMBO J. 7:1675-1682, 1988)that give position and copy number independence. The 1.5 kb Bam HI/HindIII fragment from the CD2 gene is inserted in front of gag-pol,envelope, or hybrid envelope fragments using recombinant DNA techniques.These genes are inserted into fertilized mouse ova by microinjection.Transgenic animals are characterized as before. Expression in T-cells isestablished, and animals are bred to homozygosity to establishwell-characterized lines of transgenic animals. Gag-pol animals aremated to envelope animals to establish gag-pol-env animals expressingonly in T-cells. The T-cells of these animals are then a source forT-cells capable of packaging retroviral vectors. Again, vector animalscan be bred into these gag-pol-env animals to establish T-cellsexpressing the vector.

This technique allows the use of other tissue-specific promoters, suchas milk-specific (whey), pancreatic (insulin or elastase), or neuronal(myelin basic protein) promoters. Through the use of promoters, such asmilk-specific promoters, recombinant retroviruses may be isolateddirectly from the biological fluid of the progeny.

EXAMPLE 14 Production of Either Housekeeping or Tissue-SpecificRetroviral Vectors in Transgenic Animals

The insertion of retroviruses or retroviral vectors into the germ lineof transgenic animals results in little or no expression. This effect,described by Jaenisch (see Jahner et al., Nature 298:623-628, 1982), isattributed to methylation of 5' retroviral LTR sequences. This techniquewould overcome the methylation effect by substituting either ahousekeeping or tissue-specific promoter to express the retroviralvector/retrovirus. The U3 region of the 5' LTR, which contains theenhancer elements, is replaced with regulatory sequences fromhousekeeping or tissue-specific promoters (see FIG. 20). The 3' LTR isfully retained, as it contains sequences necessary for polyadenylationof the viral RNA and integration. As the result of unique properties ofretroviral replication, the U3 region of the 5' LTR of the integratedprovirus is generated by the U3 region of the 3' LTR of the infectingvirus. Hence, the 3' is necessary, while the 5' U3 is dispensable.Substitution of the 5' LTR U3 sequences with promoters and insertioninto the germ line of transgenic animals results in lines of animalscapable of producing retroviral vector transcripts. These animals wouldthen be mated to gag-pol-env animals to generate retroviral-producinganimals (see FIG. 22).

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

We claim:
 1. A pharmaceutical composition comprising:a) a replicationdefective recombinant retrovirus which infects a human host cell, saidrecombinant retrovirus comprising a recombinant gene operable linked toa promoter, said gene encoding a protein which is not normally expressedin said host cell, and which converts a purine-based or pyrimidine-baseddrug to a compound that is toxic to said host cell, and b) apharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1 wherein said purine-based or pyrimidine-based drug is aprodrug with little or no cytotoxicity.
 3. The pharmaceuticalcomposition of claim 1 wherein said protein is guanine phosphoribosyltransferase.
 4. The pharmaceutical composition of claim 1 wherein saidprotein causes phosphorylation, ribosylation, or phosphorylribosylationof said drug.
 5. The pharmaceutical composition of claim 1 whereinexpression of said protein by said recombinant retrovirus is regulatedby the presence of an intracellular signal associated with thepathogenic state of an infectious or cancerous disease.
 6. Thepharmaceutical composition of claim 1 wherein said gene encoding saidprotein is of non-mammalian origin.
 7. The pharmaceutical composition ofclaim 1, wherein said retroviral vector also comprises a gene encoding acell surface marker.
 8. The pharmaceutical composition of claim 2wherein said protein is a nucleoside kinase.
 9. The pharmaceuticalcomposition according to claim 5 wherein said promoter is anevent-specific promoter, and wherein expression of said protein by saidpromoter is regulated by said intracellular signal.
 10. Thepharmaceutical composition of claim 5, wherein said promoter is a tissuespecific promoter.
 11. The pharmaceutical composition of claim 8 whereinsaid nucleoside kinase is a thymidine kinase.
 12. A pharmaceuticalcomposition according to claim 9 wherein said disease is a cancerousdisease and wherein said event-specific promoter is preferentiallyactive in proliferating cells.
 13. The phanmaceutical composition ofclaim 10, wherein said disease is a cancerous disease associated with atumor and wherein the tissue origin of the tissue specific promotercorresponds to the tissue origin of the tumor.
 14. The pharmaceuticalcomposition of claim 11 wherein said thymidine kinase is of viralorigin.
 15. The pharmaceutical composition of claim 13, wherein saidcancerous disease is a hepatocellular carcinoma and wherein saidtissue-specific promoter is a liver specific promoter.
 16. Thepharmaceutical composition of claim 13, wherein said recombinantretrovirus further comprises a second promoter that is an event-specificpromoter operably linked to the gene encoding said protein, and whereinthe expression of said protein is regulated by said event-specificpromoter and said tissue specific promoter.
 17. The pharmaceuticalcomposition of claim 14 wherein said thymidine kinase is herpes simplexthymidine kinase.
 18. The pbanmaeeutical composition of claim 1 or 14,wherein said human host cell is a human peripheral blood lymphocyte. 19.A pharmaceutical composition comprising:a) a producer cell whichproduces a replication defective recombinant retrovirus which infects ahuman host cell, said recombinant retrovirus comprising a recombinantgene operatively linked to a promoter, said gene encoding a proteinwhich is not normally expressed in said human host cells and whichconverts a purine-based or pyrimidine-based drug to a compound that istoxic to said human host cell; and b) a pharmaceutically acceptablecarrier.
 20. The pharmaceutical composition of claim 19, wherein saidpurine-based or pyrimidine-based drug is a prodrug with little or nocytotoxicity.
 21. The pharmaceutical composition of claim 19, whereinsaid protein is a guaniine phosphloribosyl transferase.
 22. Thepharmaceutical composition of claim 19, wherein said protein causesphosphorylation, ribosylation or phosphorylribosylation of said drug.23. The pharmaceutical composition of claim 19, wherein expression ofsaid protein by said recombinant retrovirus is regulated by the presenceof an intracellular signal associated with the pathogenic state of aninfectious or cancerous disease.
 24. The pharmaceutical composition ofclaim 19 wherein said gene encoding said protein is of non-mammalianorigin.
 25. The pharmiceutical composition of claim 19 wherein saidproducer cell is a human producler cell.
 26. The phamiaceutticalcomposition of claim 19, wherein said retroviral vector also comprises agene encoding a cell surface marker.
 27. The pharmaceutical compositionof claim 20, wherein said protein is a nucleoside kiniase.
 28. Thepharmaceutical composition of claim 23, wherein said promoter is anevent-specific promoter, and wherein expression of said protein by saidpromoter is regulated by said intracellular signal.
 29. Thepharmaceutical composition of claim 23, wherein said promoter is atissue-specific promoter.
 30. The pharmaceutical composition of claim27, wherein said nucleoside kinase is a thymidine kinase.
 31. Thepharmaceutical composition of claim 28, wherein said disease is acancerous disease and said event-specific promoter is preferentiallyactive in proliferating cells.
 32. The pharmaceutical composition ofclaim 29, wherein said disease is a cancerous disease associated withproduction of a tumor and said tissue-specific promoter has tissuespecificity that corresponds to the tissue origin of said tumor.
 33. Thepharmaceutical composition of claim 30, wherein said thymidine kinase isof viral origin.
 34. The pharmaceutical composition of claim 32, whereinsaid cancerous disease is a hepatocellular carcinoma and wherein saidtissue-specific promoter is a liver specific promoter.
 35. Thepharmaceutical composition of claim 32, wherein said recombinantretroviris further comprises a second promoter that is an event-specificpromoter operably linked to the gene encoding said protein, and whereinexpression of said protein is regulated by said event-specific promoterand said tissue-specific promoter.
 36. The pharmaceutical composition ofclaim 33, wherein said thymidine kinase is a heroes simplex thymidinekinase.
 37. A replication defective recombinant retrovirus which infectsa human host cell, said recombinant retrovirus comprising a recombinantgene operably linked to a promoter, said gene encoding a protein whichis not normally expressed in said host cell, and which converts a firstagent in said host cell to a second agent that is toxic to said hostcell.
 38. The replication defective recombinant retrovirus of claim 37wherein said first agent is a prodrug with little or no cytotoxicity.39. The replication defective recombinant retrovirus of claim 37,wherein said protein catalyzes the metabolism of a purine-based orpyrimidine-based drug.
 40. The replication defective recombinantretrovirus of claim 37, wherein expression of said protein by saidrecombinant virus is regulated by the presence of an intracellularsignal associated with the pathogenic state of an infectious orcancerous disease.
 41. The replication defective recombinant retrovirusof claim 37, wherein said gene encoding said protein is of non-mammalianorigin.
 42. The replication defective recombinant retrovirus of claim 37further comprising a gene encoding a cell surface marker.
 43. Thereplication defective recombinant retrovirus of claim 38, wherein saidprotein is a nucleoside kinase.
 44. The replication defectiverecombinant retrovirus of claim 39, wherein said protein is guaninephosphoribosyl transferase.
 45. The replication defective recombinantretrovirus of claim 39, wherein said protein causes phosphorylation,ribosylation, or phosphoribosylation of said drug.
 46. The replicationdefective recombinant retrovirus of claim 37 or 39, wherein said humanhost cell is a human peripheral blood lymphocyte.
 47. The replicationdefective recombinant retrovirus of claim 40, wherein said promoter isan event-specific promoter, and wherein expression of said protein bysaid promoter is regulated by said intracellular signal.
 48. Thereplication defective recombinant retrovirus of claim 40, wherein saidpromoter is a tissue specific promoter.
 49. The replication defectiverecombinant retrovirus of claim 43, wherein said nucleoside kinase is athymidine kinase.
 50. The replication defective recombinant retrovirusaccording to claim 47, wherein said disease is a cancerous disease andwherein said event-specific promoter is preferentially active inproliferating cells.
 51. The replication defective recombinantretrovirus of claim 48, wherein said disease is a cancerous diseaseassociated with a tumor and wherein the tissue origin of the tissuespecific promoter corresponds to the tissue origin of the tumor.
 52. Thereplication defective recombinant retrovirus of claim 49, wherein saidthymidine kinase is herpes simplex thymidine kinase.
 53. The replicationdefective recombinant retrovirus of claim 51, wherein said cancerousdisease is hepatocellular carcinoma and wherein said tissue-specificpromoter is a liver-specific promoter.
 54. The replication defectiverecombinant retrovirus of claim 51, wherein said recombinant retrovirusfurther comprises a second promoter that is an evenit-specific promoteroperably linked to the gene encoding said protein, and wherein theexpression of said protein is regulated by said event-specific promoterand said tissue specific promoter.
 55. A producer cell which produces areplication defective recombinant retrovirus which infects a human hostcell, said recombinant retrovirus comprising a recombinant gene operablylinked to a promoter, said gene encoding a protein not normallyexpressed in said human cell, and which converts an agent in said humanhost cell to a second agent that is toxic to said human host cell. 56.The producer cell of claim 55, wherein said first agent is a prodrugwith little or no cytotoxicity.
 57. The producer cell of claim 55,wherein said protein catalyzes the metabolism of a purine-based orpyrimidine-based dnig.
 58. The producer cell of claim 55, whereinexpression of said protein by said recombinant retrovirus is regulatedby the presence of an intracellular signal associated with thepathogenic state of an infectious cancerous disease.
 59. The producercell of claim 55, wherein said gene encoding protein is of non-mammalianorigin.
 60. The producer cell of claim 55, wherein the producer cell isa human producer cell.
 61. The producer cell of claim 55, wherein saidrecombinant retrovirus further comprises a gene encoding a cell surfacemarker.
 62. The producer cell of claim 56, wherein said protein is anucleoside kinase.
 63. The producer cell of claim 57, wherein saidprotein is a guanine phosphoribosyl transferase.
 64. The producer cellof claim 57, wherein said protein causes phosphorylation, ribosylationor phosphorylribosylation of said drug.
 65. The producer cell of claim58, wherein said promoter is an event-specific promoter, and whereinexpression of said protein by said promoter is regulated by saidintracellular signal.
 66. The producer cell of claim 58, wherein saidpromoter is a tissue-specific promoter.
 67. The producer cell of claim62, wherein said nucleoside kinase is a thymidine kinase.
 68. Theproducer cell of claim 65, wherein said disease is a cancerous diseaseand said event-specific promoter is preferentially active inproliferating cells.
 69. The producer cell of claim 66, wherein saiddisease is a cancerous disease associated with production of a tumor andsaid tissue-specific promoter has tissue specificity that corresponds tothe tissue origin of said tumor.
 70. The producer cell of claim 67,wherein said thymidine kinase is a herpes simplex thymidine kinase. 71.The producer cell of claim 69, wherein said cancerous disease is ahepatocelluilar carcinoma and wherein said tissue-specific promoter is aliver specific promoter.
 72. The producer cell of claiim 69, whereinsaid recombinant retrovirus further comprises a second promoter that isan event-specific promoter operably linked to the gene encoding saidprotein, and wherein expression of said protein is regulated by saidevent-specific promoter and said tissue-specific promoter.